Neutralizing antibodies to hiv-1 and their use

ABSTRACT

Monoclonal neutralizing antibodies are disclosed that specifically bind to the CD4 binding site of HIV-1 gp120. Monoclonal neutralizing antibodies also are disclosed that specifically bind to HIV-1 gp41. The identification of these antibodies, and uses of these antibodies, are also disclosed. Methods are also provided for enhancing the binding and neutralizing activity of any antibody using epitope scaffold probes.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of International Application No.PCT/US2010/050295, filed Sep. 24, 2010, which in turn claims the benefitof U.S. Provisional Application No. 61/385,531, filed Sep. 22, 2010,U.S. Provisional Application No. 61/402,314, filed Aug. 27, 2010, U.S.Provisional Application No. 61/346,808, filed May 20, 2010, U.S.Provisional Application No. 61/290,135, filed Dec. 24, 2009, U.S.Provisional Application No. 61/252,613, filed Oct. 16, 2009, and U.S.Provisional Application No. 61/246,039, filed Sep. 25, 2009. All of theprior applications are specifically incorporated by reference in theirentirety.

FIELD OF THE DISCLOSURE

This relates to monoclonal neutralizing antibodies that bind to the CD4binding site of HIV-1 gp120 or to HIV-1 gp41, their identification, andtheir use.

BACKGROUND

An effective HIV-1 vaccine will likely need to induce neutralizingantibodies (NAbs) that block HIV-1 entry into human cells. To beeffective, vaccine induced antibodies will have to be active againstmost circulating strains of HIV-1. Unfortunately, current HIV-1 vaccinesare unable to induce potent and broadly reactive NAbs. One majorobstacle to the design of better vaccines is the limited understandingof what region of the HIV-1 envelope glycoproteins (gp120 and gp41) arerecognized by NAbs. A few neutralizing monoclonal antibodies (mAbs) havebeen isolated from HIV-1 infected individuals and these mAbs definespecific regions (epitopes) on the virus that are vulnerable to NAbs.

One previously characterized HIV-1 neutralizing mAb, called b12, canbind to a site on gp120 that is required for viral attachment to itsprimary cellular receptor, CD4. Another previously characterized HIV-1neutralizing mAb, called 2F5, can bind to a site on gp41. mAb b12 wasderived from a phage display library, a process which makes itimpossible to know if the antibody was naturally present in an infectedperson, or was the result of a laboratory combination of antibody heavyand light chains. b12 can neutralize about 75% of clade B strains ofHIV-1 (those most common in North America), but it neutralizes less than50% of other strains of HIV-1 found worldwide. Prior attempts to designa vaccine that induces NAbs similar to b12 have been unsuccessful.Therefore, there is a need to develop Nabs for HIV-1.

SUMMARY OF THE DISCLOSURE

Isolated human monoclonal neutralizing antibodies that specifically bindHIV-1 gp120 or gp41 are provided herein. Also disclosed herein arecompositions including these antibodies that specifically bind gp120- orgp41-nucleic acids encoding these antibodies, expression vectorscomprising the nucleic acids, and isolated host cells that express thenucleic acids. In some embodiments, the heavy chain of the isolatedhuman monoclonal antibody includes amino acids 26-33(complementarity-determining region 1 (CDR1)), 51-58 (CDR2), and 97-110(CDR3) of SEQ ID NO: 1, wherein the antibody specifically binds gp120 ofHIV-1, and wherein the antibody is neutralizing. In other embodiments,the heavy chain of the isolated human monoclonal antibody includes aminoacids 26-35 (CDR1), 50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO: 27,wherein the antibody specifically binds gp120 of HIV-1, and wherein theantibody is neutralizing. In other embodiments, the heavy chain of theisolated human monoclonal antibody includes CDR1, CDR2, and CDR3 of anyone of SEQ ID NOs: 760-1459, wherein the antibody specifically bindsgp120 of HIV-1, and wherein the antibody is neutralizing. In still otherembodiments, the heavy chain of the antibody includes SEQ ID NO: 5,wherein one or more of amino acids 106, 107, or 109 of SEQ ID NO: 5 aresubstituted with a tryptophan, and wherein the antibody specificallybinds gp41 of HIV-1 and is neutralizing.

The antibodies and compositions disclosed herein can be used for avariety of purposes, such as for detecting an HIV-1 infection ordiagnosing AIDS in a subject. These methods can include contacting asample from the subject diagnosed with HIV-1 or AIDS with a humanmonoclonal antibody that specifically binds gp120 or gp41, and detectingbinding of the antibody to the sample. An increase in binding of theantibody to the sample relative to binding of the antibody to a controlsample confirms that the subject has an HIV-1 infection and/or AIDS. Insome embodiments, the methods further comprise contacting a secondantibody that specifically binds gp120 or gp41 with the sample, anddetecting binding of the second antibody. In some non-limiting examplesan increase in binding of the antibody to the sample relative to acontrol sample detects HIV-1 in the subject. In some non-limitingexamples, the antibody specifically binds soluble gp120 in the sample.In some embodiments, the methods further comprise contacting a secondantibody that specifically recognizes the 120- or gp41-specific antibodywith the sample and detecting binding of the second antibody.

In additional embodiments, a method is disclosed for treating a subjectwith an HIV infection, such as, but not limited to, a subject with AIDS.The methods include administering a therapeutically effective amount ofa human gp120 or gp41 specific monoclonal antibody to the subject.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of aseveral embodiments which proceeds with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E are a set of surface representations and graphs showing thedesign and antigenic profile of RSC3 and analysis of epitope-specificneutralization. FIG. 1A is a surface structure model of the RSC3. Theouter domain contact site for CD4 is highlighted in light grey. Regionshighlighted dark are antigenically resurfaced areas, shown both on theinner (left panel) and outer (right panel) faces of the core protein.Glycans are shown in medium grey. FIG. 1B is a set of graphs showing theantigenicity of the RSC3 protein based on enzyme-linked immunosorbentassay (ELISA) using the neutralizing CD4bs mAb b12 and CD4-Ig fusionprotein. mAb 2G12 was used to confirm the structural integrity of theprotein. FIG. 1C is a set of graphs showing the results of kineticbinding analysis in which mAb b12 was immobilized on the sensor chip forsurface plasmon resonance (SPR) kinetic binding analysis with theproteins shown. FIG. 1D is a set of graphs showing the RSC3 blockade ofHIV-1 viral strain HXB2 neutralization by the broadly neutralizing CD4bsmAb b12, but not CD4bs mAb F105, which has limited neutralizationbreadth. The V3 neutralizing mAb 447-52D is shown as a control. FIG. 1Eis a set of bar graphs showing the analysis of serum 45 neutralizationof a panel of 17 viruses, using RSC3 and ΔRSC3 to block neutralizationactivity. The percent reduction in the serum ID50 caused by competitionwith RSC3 or ΔRSC3 is shown on the Y-axis (+/−SEM of three independentexperiments). Viral strains and clades are shown on the X-axis. Valuesless than 20% were not considered significant in this assay.

FIGS. 2A-2C are a set of dot plots and graphs showing isolation ofindividual CD4bs-directed memory B cells by cell sorting, and bindingcharacterization of isolated mAbs. FIG. 2A is a set of dot plots of flowcytometry data showing the results of twenty-five million peripheralblood mononuclear cells (PBMC) from donor 45 that were incubated withbiotin-labeled RSC3 and ΔRSC3 complexed with SA-APC and SA-PErespectively, prior to addition to cells. Memory B cells were selectedbased on the presented gating strategy. Twenty-nine B cells that reactedwith RSC3 and not ΔRSC3 (representing 0.05% of all memory B cells) weresorted into individual wells of a 96 well plate containing lysis buffer.FIG. 2B is a set of graphs of the results of an ELISA antigen bindingprofile of three isolated mAbs, VRC01, VRC02 and VRC03. Solid lines showmAb binding to RSC3 (left panel) and YU2 gp120 (right panel). Dashedlines indicate binding to ΔRSC3 (left) or to the CD4bs knockout mutantof gp120, D368R (right). FIG. 2C is a set of graphs of the SPR bindinganalysis of VRC01 reacted with RSC3 and ΔRSC3. VRC01 was captured withan anti-human IgG-Fc antibody that was immobilized on the sensor chip.

FIGS. 3A and 3B are a set of graphs and a table showing the antigenicand biophysical characterization of novel CD4bs-directed mAbs. FIG. 3Ais a set of graphs of results of competition ELISA performed with asingle concentration of biotin-labeled VRC01 (left) or the co-receptorbinding mAb 17b (right). The mAbs indicated near each line were titratedinto the ELISA at increasing concentrations to evaluate the effect onVRC01 and 17b binding, respectively. FIG. 3B is a table showing theresults of isothermal titration calorimetry (ITC) used to assess thechange in enthalpy (ΔH) and entropy (−TΔS) upon binding of mAbs to YU2gp120. Each measured value is shown +/−SEM.

FIG. 4 is set phylogenetic trees showing the analysis of neutralizationby mAbs VRC01 and b12 against a panel of 190 Env pseudovirusesrepresenting all major circulating clades of HIV-1. Dendrograms, made bythe neighbor-joining method, show the protein distance of gp160sequences from 190 HIV-1 primary isolates. The clade B reference strainHXB2 was used to root the tree, and the amino acid distance scale isindicated with a value of 1% distance as shown. The clades of HIV-1 maingroup, including circulating recombinant forms (CRFs), are indicated.The data under the dendrograms show the percent of viruses neutralizedwith an IC50<50 μg/ml, and <1 μg/ml, and the geometric mean IC50 valuefor viruses neutralized with an IC50<50 μg/ml.

FIG. 5A is a schematic drawing showing an algorithm of structure-baseddesign of the resurfaced core proteins. The design intent was toresurface non-CD4bs regions of the core protein, and to maintain the b12contact surface while abrogating CD4 binding.

FIG. 5B is a protein sequence alignment of the resurfaced proteins andthe HIV-1 HXB2 core or stabilized core (Ds12F123), which provided theframework for the resurfaced protein designs. Residue positions aremarked according to the HXB2 sequence. Highlighted are amino acidsubstitutions made in the resurfaced proteins in comparison to theoriginal HXB2 core or stabilized core sequences. Gaps are indicated as“-”.

FIG. 6 is a table showing the design and expression of resurfaced core(RC) and resurfaced stabilized core (RSC) glycoproteins, and summary ofantigenic reactivity. A panel of 8 resurfaced proteins with differentdegrees of resurfacing were designed and tested. The surface structuralmodel of each resurfaced protein is shown. Each protein was analyzed forbinding to CD4-Ig, b12 and 2G12. 2G12 binding was used as a marker ofconformational integrity of the purified protein. ELISA binding activitywas categorized as strong (+++), moderate (++), weak (+) or negative(−). RSC3, used subsequently to isolate mAbs, was 31.3% antigenicallyresurfaced and maintained strong binding to b12.

FIG. 7 is a table showing the results of binding to RSC3 protein by apanel of neutralizing sera. Fifteen clade B sera with moderate to broadneutralizing activity were evaluated by ELISA for binding to RSC3 andΔRSC3. Neutralization of five viral isolates is shown; the clade of eachvirus is indicated in parenthesis. Neutralization ID50 values greaterthan 1000 are highlighted in dark boxes; values between 100-1000 arehighlighted in light boxes. ELISA binding was categorized as strong(+++), moderate (++), weak (+) or negative (−). Preferential binding toRSC3, compared to ΔRSC3, is evidence of CD4bs directed antibodies in thesera. Donor 45 was chosen for additional serum analysis, and eventuallyfor isolation of mAbs.

FIG. 8 is a set of graphs showing the comparison of VRC01 and b12binding kinetics by surface plasmon resonance (SPR). The mAbs werecaptured with a mouse anti-human IgG Fc antibody that was immobilizedonto the chip matrix. The binding kinetics of ligands RSC3, ΔRSC3,stabilized core and full-length YU2 gp120 were analyzed.

FIG. 9 is a set of graphs showing the VRC02 and VRC03 binding kineticsby SPR. The mAbs were captured with a mouse anti-human IgG Fc antibodythat was immobilized onto the chip matrix. The binding kinetics ofligands RSC3, ΔRSC3, stabilized core and full-length YU2 gp120 wereanalyzed.

FIG. 10A is a table showing a gene family analysis of VRC01, VRC02,VRC03 and b12. The VH and VK mutation frequency was calculated from themutated nucleotides. Mean values from three normal donors consisted of120 IgD+CD27+ and 97 IgDCD27+ sequences for heavy chain analysis and 167mutated IgM+ sequences for kappa analysis are shown. A specific D genecould not be determined since the germline genes with the greatesthomology (IGHD3/OR15-3, IGHD3-22 or IGHD3-16) each contained a mutationwithin a matching length of less than 11 nucleotides and the orphanIGHD3/OR15-3 gene on chromosome 15 cannot contribute to Ig chainsynthesis; bIGHJ2*01 is an alternative possibility based on the thirdcomplementarity determining region 3 (CDR3) sequence analysis;cIGKV3-NL1*01 (NL=Not Located) showed greater homology than IGKV3-11*01by one nucleotide; dIGKV3-NL5*01 showed greater homology thanIGKV3-20*01 by one nucleotide.

FIG. 10B is sequence alignment of the deduced amino acid sequences ofthe variable regions of VRC01 (heavy chain, SEQ ID NO: 1; light chain,SEQ ID NO: 2), VRC02 (heavy chain, SEQ ID NO: 3; light chain, SEQ ID NO:4) and VRC03 (heavy chain, SEQ ID NO: 27; light chain, SEQ ID NO: 28).Framework (FR) and CDRs are indicated above the sequence alignment. Thetop sequence in each group represents the deduced germline sequence withidentity to the expressed VH1, D3, JH1, VK3 and JK2 genes. VRC01, VRC02and VRC03 were derived from the same VH germline gene (IGHV1-02*02, SEQID NOs: 41, 42), hence all 3 mAbs are aligned to this sequence. VRC01and VRC02 are somatic variants of each other (they have the same V-D-Jrecombination). The arrow marks the position of a 7 amino acidsinsertion (QLSQDPD, SEQ ID NO: 27) in the VRC03 heavy chain FR3 region.A common motif is underlined in the heavy chain CDR1 (GYXFXD, SEQ ID NO:27), CDR2 (KPXXGAV, SEQ ID NO: 27) and CDR3 (CDYXXDF, SEQ ID NO: 27).VRC01 and VRC02 have the same VK gene (IGKV3-11*01, SEQ ID NO: 42).While the closest inferred germline sequence match for VRC03 wasIGKV3-20*01 (SEQ ID NO: 43), IGKV3-11*01 was also a close match. The dotsymbol marks an amino acid deletion in the VK CDR1.

The arrows mark the position of a 2 amino acids insertion (VQ) in VRC01and VRC02 FR4 (JK gene). Light residues indicate replacementsubstitutions compared to germline sequence.

FIG. 11A is two graphs showing the results of competition ELISAperformed with a single concentration (50 ng/ml) of biotinylated VRC02or VRC03 binding to YU2 gp120. The unlabeled competing mAbs weretitrated into the ELISA at increasing concentrations to evaluate theeffect on VRC02 and VRC03 binding respectively.

FIG. 11B are three graphs showing the results of competition ELISAperformed with a single concentration (50 ng/ml) of biotinylated VRC01,VRC02 or VRC03 binding to RSC3. The unlabeled competing mAbs werelimited to those that showed binding to RSC3.

FIG. 11C is a graph showing the results of analysis of gp120 binding tocell surface expressed CCR5 by flow cytometry. Biotinylated gp120 at 5μg/ml was used to stain the human CCR5 expressing canine thymus cellline, Cf2Th/syn CCR5. Prior to cell staining, biotinylated gp120 wasincubated with CD4-Ig or mAbs VRC01, VRC03 or b12 with serialconcentrations ranging from 0.04-25 μg/ml. Binding of gp120 was detectedby streptavidin-APC and FACS analysis. Note that CD4-Ig and VRC01enhanced gp120 binding to CCR5, while mAbs b12 and VRC03 did not.

FIGS. 12A and 12B are a set of graphs showing analysis of the effect ofVRC01 on the functional viral spike. FIG. 12A is a set of graphs showingthe neutralization of JRFL that was performed with the mAbs shown in thelegend. Antibody 447-52D is directed to the V3 region of gp120, and mAbs17b and vc813 DB are directed to the co-receptor binding region ofgp120. Graphs show the effect on neutralization as sCD4 (left panel) orVRC01 (right panel) were added to the assay. The adjusted neutralizationwas calculated using the baseline of viral entry at each concentrationof sCD4 or VRC01. In contrast to sCD4, VRC01 did not enhance theneutralization by mAbs 447-52D, 17b and vc813 DB. FIG. 12B is a set ofgraphs showing JRFL entry into the CCR5+/CD4-cell line, Cf2Th/syn CCR5.CD4-Ig and sCD4 (left panel) promote entry of JRFL into CD4 negativecells. VRC01 (right panel) did not promote viral entry. Each infectionwas performed in triplicate, and the mean and standard error are shown.

FIGS. 13A and 13B are a table and a graph showing correlation analysisof neutralization by serum 45 IgG and mAb VRC01. FIG. 13A is acontingency table showing neutralization by serum 45 IgG and mAb VRC01.Fisher's exact test demonstrated a strong association between the numberof viruses neutralized by serum 45 IgG and mAb VRC01. Serum 45 IgGsensitive was defined as an IC50<1000 μg/ml. VRC01 sensitivity wasdefined as an IC50 <50 μg/ml. FIG. 13B is a graph of a deming regressionanalysis of log transformed IC50 values of viruses neutralized by bothVRC01 and serum 45 IgG. This showed a strong association between thepotency of serum 45 IgG and mAb VRC01. The slope of the regression lineis 0.68 (95% CI 0.07). Thus, while VRC01 accounts for a substantialportion of total serum 45 IgG neutralization, the slope of less than 1.0suggests that VRC01 does not account for all serum 45 IgG neutralizationactivity.

FIG. 14 is Table S1 showing the ELISA binding profiles of VRC01, VRC02,and VRC03 compared to a panel of known mAbs.

FIG. 15 is Table S2a showing a summary of the breath and potency ofantibody neutralization against 190 HIV-1 Env-pseudoviruses.

FIG. 16 is Table S2b showing antibody neutralization data against 22HIV-1 clade A HIV-1 Env-pseudoviruses.

FIG. 17 is Table S2c showing antibody neutralization data against 49HIV-1 clade B HIV-1 Env-pseudoviruses.

FIG. 18 is Table S2d showing antibody neutralization data against 38HIV-1 clade C HIV-1 Env-pseudoviruses.

FIG. 19 is Table S2e showing antibody neutralization data against 8HIV-1 clade D HIV-1 Env-pseudoviruses.

FIG. 20 is Table S2f showing antibody neutralization data against 18HIV-1 CRF01_AE Env-pseudoviruses.

FIG. 21 is Table S2g showing antibody neutralization data against 16HIV-1 CRF02_AG Env-pseudoviruses.

FIG. 22 is Table S2h showing antibody neutralization data against 10HIV-1 clade G Env-pseudoviruses.

FIG. 23 is Table S21 showing antibody neutralization data against 11HIV-1 CRF07_BC Env-pseudoviruses.

FIG. 24 is Table S2j showing antibody neutralization data against 18Env-pseudoviruses.

FIG. 25 is Table S3 showing IC50 titers (μg/ml) of antibodyneutralization against selected HIV-1 clade B and C viruses usingEnv-pseudoviruses to infect TZM-bl or activated PBMC, and using PBMCderived uncloned primary isolates to infect TZM-bl or activated PBMC.

FIG. 26 is an electronic image of a depiction of the structure ofantibody VRC01 in complex with HIV-1 gp120. Atomic-level details forbroad and potent recognition of HIV-1 by a natural human antibody aredepicted with polypeptide chains in ribbon representations. Both lightand heavy chains of VRC01 interact with gp120: the primary interactionsurface is provided by the CDR H2, with the CDR L1 and L3 and the CDR H1and H3 providing additional contacts.

FIGS. 27A-27D are a set of electronic images depicting the structuralmimicry of CD4 interaction by antibody VRC01. VRC01 shows how adouble-headed antibody can mimic the interactions with HIV-1 gp120 of asingle-headed member of the immunoglobulin superfamily such as CD4. FIG.27A is a comparison of HIV-1 gp120 binding to CD4 (N-terminal domain)and VRC01 (heavy chain-variable domain). Polypeptide chains are depictedin ribbon representation for the VRC01 complex (right) and the CD4complex with the lowest gp120 RMSD (left). Immunoglobulin domains arecomposed of two β-sheets, and the top sheet of both ligands is labeledwith the standard immunoglobulin-strand topology (strands G, F, C, C′,C″). Close-ups are shown of critical interactions between theCD4-binding loop and the C′″ strand as well as between Asp368gp120 andeither Arg59CD4 or Arg71VRC01. Atoms from which hydrogen bonds extendare depicted in stick representation. In the left panel of C, theβ15-strand of gp120 is depicted to aid comparison with FIG. 27B, thoughbecause of the poor hydrogen-bond geometry, it is only a loop. FIG. 27Dshows the comparison of VRC01- and CD4-binding orientations. When theheavy chain of VRC01 is superimposed onto CD4 in the CD4-gp120 complex,the position assumed by the light chain evinces numerous clashes withgp120 (left). The VRC01— binding orientation (right) avoids clashes byadopting an orientation rotated by 43° and translated by 6-Å.

FIGS. 28A-28C are a dendrogram, a bar graph, and a set of graphs showingstructural basis of antibody VRC01 neutralization breadth and potency.VRC01 displays remarkable neutralization breadth and potency, aconsequence in part of its ability to bind well to differentconformations of HIV-1 gp120. FIG. 28A shows neutralization dendrograms.The genetic diversity of current circulating HIV-1 strains is displayedas a dendrogram, with locations of prominent clades (e.g. A, B and C)and recombinants (e.g., CDR02_AG) labeled. VRC01 neutralizes 72% of thetested HIV-1 isolates with an IC80 of less than 1 ug/ml; by contrast,CD4 neutralizes 30% of the tested HIV-1 isolates with an IC80 of lessthan 1 ug/ml. FIG. 28B shows a comparison of binding affinities. Bindingaffinities (KDs) for VRC01 and various other gp120-reactive ligands asdetermined by surface-plasmon resonance are shown on a bar graph. Whitebars represent affinities for gp120 restrained from assuming theCD4-bound state and black bars represent affinities for gp120 fixed inthe CD4-bound state. Binding too weak to be measured accurately is shownas with an asterisk and bar at 10⁻⁵ M KD. FIG. 28C shows theneutralization of viruses with altered sampling of the CD4-bound state.Mutant S375Wgp120 favors the CD4-bound state, whereas mutants H66Agp120and W69Lgp120 disfavor this state. Neutralization by VRC01 (left) issimilar for wild-type (WT) and all three mutant viruses, whereasneutralization by CD4 (right) correlates with the degree to which gp120in the mutant viruses favors the CD4-bound state.

FIGS. 29A-29D are electronic images depicting the natural resistance toantibody VRC01. VRC01 precisely targets the CD4-defined site ofvulnerability on HIV-1 gp120. Its binding surface, however, extendsoutside of the target site, and this allows for natural resistance toVRC01 neutralization. FIG. 29A shows the target site of vulnerability.The CD4-defined site of vulnerability is the initial contact surface ofthe outer domain of gp120 for CD4 and comprises only two-thirds of thecontact surface of gp120 for CD4. The view shown here is rotated 90°about the horizontal from the view in FIGS. 26 and 27. FIG. 29B showsVRC01 recognition. The variable domains of VRC01 are shown in ribbonrepresentation. FIG. 29C shows antigenic variation. FIG. 29D, shows amolecular surface of VRC01 and select interactive loops of gp120.Variation at the tip of the V5 loop is accommodated by a gap betweenheavy and light chains of VRC01.

FIGS. 30A-30D are a set of electronic images, bar graphs and graphsdepicting the unusual VRC01 features. The structure of VRC01 displays anumber of unusual features, which if essential for recognition mightinhibit the elicitation of VRC01-like antibodies. In FIG. 30A-30D,unusual features of VRC01 are shown structurally (far left panel), interms of frequency as a histogram with other antibodies (second panelfrom left), and in the context of affinity and neutralizationmeasurements after mutational alteration (right two panels). Affinitymeasurements were made by ELISA to the gp120 construct used incrystallization (93TH057), and neutralization measurements were madewith a clade A HIV-1 strain Q842.d12. FIG. 30A shows N-linkedglycosylation. The conserved tri-mannose core is shown with observedelectron density, along with frequency and effect of removal onaffinity. FIG. 30B shows an extra disulfide. Variable heavy domainsnaturally have two Cys, linked by a disulfide; VRC01 has an extradisulfide linking CDR H1 and H3 regions. This occurs rarely inantibodies, but its removal by mutation to Ser/Ala has little effect onaffinity. FIG. 30C shows CDR L1 deletion. A two amino acid deletion inthe CDR L1, prevents potential clashes with loop D of gp120. Suchdeletions are rarely observed; reversion to the longer loop may have a10-100-fold effect on gp120 affinity. FIG. 30D shows the somaticallyaltered contact surface. The far left panel shows the VRC01 light chainand heavy chain. Residues altered by affinity maturation are depictedwith “balls” and contacts with HIV-1 gp120 are colored red. About halfthe contacts are altered during the maturation process. Analysis ofhuman antibody-protein complexes in the protein-data bank shows thisdegree of contact surface alteration is rare; reversion of each of thecontact site to genome has little effect though in aggregate the effecton affinity is larger.

FIGS. 31A-31B are a set of graphs showing somatic maturation and VRC01affinity. Hypermutation of the variable domain during B cell maturationallows for the evolution of high affinity antibodies. With VRC01 thisenhancement to affinity occurs principally through the alteration ofnon-contact residues, which appear to reform the genomic contact surfacefrom affinity too low to measure to a tight (nM) interaction. FIG. 31Ashows the effect of genomic reversions. The VH- and VK-derived regionsof VRC01 were reverted to the sequences of their closest genomicprecursors, expressed as immunoglobulins and tested for binding as VH-and VK-revertants (gHgL), as a VH-only revertant (gH), or as a VK-onlyrevertant (gL) to the gp120 construct used in crystallization (93TH057)or to a stabilized HXBc2 core. These constructs were also tested forneutralization of a clade A HIV-1 strain Q842.d12. FIG. 31B showsmaturation of VRC01 and correlation with binding and neutralization.Affinity and neutralization measurements for the 19 VRC01 mutantscreated during the structure-function analysis of VRC were analyzed inthe context of their degree of affinity maturation. Significantcorrelations were observed, with extrapolation to VH- and VK-genomicrevertants suggesting greatly reduced affinity for gp120.

FIG. 32 is a gp120 sequence alignment and residue-by-residue contactswith CD4 and VRC01. Both wild type clade B HXBc2 (SEQ ID NO: 44) andclade A/E 93TH057 core gp120 sequences (SEQ ID NO: 45) are displayedwith HXBc2 numbering convention. The 93TH057 construct has shorter V1/V2stem and has a new V3 stem. gp120 contacts are defined with the programPISA for the CD4 and VRC01 complexes, with open circles (◯)denotinggp120 main-chain-only contacts, open circles with rays (¤)denoting gp120side-chain-only contacts, and filled circles () denoting bothmain-chain and side-chain contacts. The major structure elements ofgp120 that involved in ligand binding were underlined. Potentialglycosylation sites on gp120 with signature sequence NXT/S arehighlighted in cyan, however, not all sites are observed in the crystalstructure. VRC01 has remarkably less interactions with theconformationally variable V1/V2 and β20/β21 regions and moreinteractions at the loop D and V5 areas.

FIG. 33 is a set of electronic images showing electrostatic surfaces andmaps of residues types of gp120. Electrostatic surfaces for the VRC01and CD-bound gp120s are shown in the left panels with heavy chainvariable domain (VH) of VRC01 (upper row) and domain 1 (D1) of CD4(lower row). Both of VRC01 and CD4 bind to overall negatively chargedsurfaces on gp120. The flip sides of the complexes showing theelectrostatic surfaces of VH and D1 are presented in the middle panelswith gp120 in the foreground. The gp120 interfaces on VH of VRC01 and D1of CD4 are mostly positively charged to complement the negativelycharged gp120 surfaces. Certain residues, such as Arg71 in VRC01 andArg59 in CD4, are conserved, the unique VRC01 Årg61 that penetrating thecavity formed by V5 and (324 is also shown. The electrostatic potentialwas calculated with APBS and visualized with Pymol.

FIG. 34 is a set of graphs showing the binding of VRC01 to gp120stabilized in CD4-bound and non-CD4-bound conformations. Both gp120 innon-CD4-bound conformation (YU2 Δβ4) and gp120 stabilized in CD4-boundconformation (HXBc2 core Ds12F123) were immobilized on a CM-5 chip. Fabsof CD4-binding site antibodies VRC01, b12 and F105 and CD4-inducedantibody 17b, two domain CD4 and CD4-IgαTP at various concentrationswere injected over the chip channels.

FIGS. 35A and 35B are a set of electronic images and a bar graph showinga comparison of coverage of the site of vulnerability by differentCD4-binding site antibodies. The site of vulnerability is the contactsite for receptor CD4 on the outer domain of gp120.CD4-binding-site-directed antibodies target this general area, however,most of them do not neutralize potently. FIG. 35A shows that when thesite of vulnerability is superimposed over the antibody epitopes ongp120 surfaces, the degrees of overlapping differ. VRC01 hits the“bull's-eye” while b12, b13 and F105 miss portions of the target withepitope straying away to other conformationally variable areas on gp120.FIG. 35B shows that when coverage of the site of vulnerability byepitopes of CD4-binding-site-directed antibodies were compared, VRC01achieves almost full coverage (98%) while others, such as b13, F105 andb12, manage to get 50% to 83% overlapping coverage with CD4.

FIGS. 36A-36D are a set of electronic images and a sequence alignmentshowing the mechanism of natural resistance to VRC01. FIG. 36A shows asequence threading of the 17 HIV-1 isolates that resist neutralizationby VRC01. Spots that are closer than 2.5 Å to VRC01 are dark. Thesespots are clustered at the loop D and V5 region on HIV-1gp120. FIG. 36Bshows a close-up of threaded, resistant isolates along with themolecular surface of VRC01. FIG. 36C shows VRC01 heavy chain Arg61penetrating the gp120 cavity formed by V5 and (324. Some resistantisolates have bulky residues pointing into the cavity which interferewith Arg61VRC01 without affecting CD4 binding. FIG. 36D shows a sequencealignment of VRC01-resistant isolates at the V5 region (SEQ ID NO: 46).Black boxes highlight bulky residues that may interfere with binding ofVRC01 and are different from the 93TH057 sequence. Different N-linkedglycosylation patterns are also marked.

FIG. 37 is an electronic image showing the conformational variation ofgp120 loopV5. Side-by-side comparison of conformation variation at theHIV-1 gp120 variable loop 5 region indicates that the four gp120components (left panel, chains G, A, D and I) of the VRC01:gp120complexes in the crystallographic asymmetric unit vary only at the tipof V5 loop and conformation of the V5 base is less flexible due toincreased contacts by VRC01. In contrast, variation of V5 conformationsin other gp120 complexes (right panel) with CD4 and CD4-binding siteantibodies, F105, b12 and b13, spans over the whole range of V5 loop.

FIGS. 38A-38B are electronic images showing that VRC01 recognition of V5and β24 of gp120 is different from that of CD4. FIGS. 38A and 38Billustrate that the V5 loop is wedged in the gap formed by the heavy andlight chains of VRC01, meanwhile, Arg61 in the CDRH2 penetrates into thecavity formed by gp120 V5 and β24, locking V5 into a less flexibleconformation. In contrast, CD4 only interacts with the “front side” ofV5. FIGS. 38C and 38D illustrates that VRC01 engages extensiveinteractions with V5 and (324 with 10 hydrogen bonds and a salt bridgefrom both CDR H2 and CDR L3. While heavy chain Asn68, G1n64 and lightchain Glu96 grab the front side of V5, heavy chain Arg61 goes behind theV5 and provides 4 hydrogen bonds to residues on β24. CD4, however, onlyhas 3 hydrogen bonds to 2 V5 residues. It is worth to note that VRC01only interacts with residues at the base of V5 loop and avoids the looptip which has higher degree of sequence variation. The VRC01 CDRs areshown. Selected gp120 residues are labeled in italic.

FIG. 39 is a set of electronic images showing the key interface regionsof the gp120:VRC01 complex. gp120-interacting CDRs of VRC01 areprojected over the gp120 surface (left panel). Both heavy and lightchains are involved in binding of gp120, mainly to the conformationallyinvariant outer domain. The CDR H2 spans over the CD4-binding loop andthe V5/β24, with Arg61 penetrating the V5/β24 cavity (right panel).Arg71 in the framework 3 forms salt bridges with a conserved Asp368 inthe CD4-binding loop of gp120. The light chain of CDRL1 and CDRL3provide interactions to V5, loop D as well as the Loop D attachedN-acetyl-glucosamine of a N-linked glycan.

FIG. 40 is an alignment of the VRC01 sequence, gp120 contacting-sitesand extent of affinity maturation. The sequence of VRC01 (SEQ ID NO: 1and SEQ ID NO: 2) is shown along with nearest VH- and VK/λ-genomicprecursors for heavy (SEQ ID NO: 41) and light chain (SEQ ID 42),respectively. Affinity maturation changes are indicated in green, withresidues involved in interaction with HIV-1 gp120 highlighted by “”, ifinvolved in both main- and side-chain interactions, by “◯” if mainchain-only, and by “¤” if side chain-only. “

” marks a site of N-linked glycosylation, “▴” for Cysteine residuesinvolved in a noncanonical disulfide, and “Δ” if the residue has beendeleted during affinity maturation.

FIG. 41 is a bar graph showing the contact, V-gene mutated, and mutatedcontact residues for the set of 26 antibodies and VRC01. Any antibodyresidue in contact with the antigen in the complex is included towardsthe total number of contact residues. The number of mutations fromgermline for the Vh and Vl/Vk, as well as the number of mutated contactresidues are shown. The number of mutations excludes insertions anddeletions. Germline alignment was performed using the amino acidsequences for the antibody heavy and light chains. For comparison, alsoshown is the number of mutated residues (total and only contactresidues) when germline alignment is performed for the **VRC01nucleotide sequence.

FIG. 42 is an electronic image showing of the contributions of VRC01VH-D-J and VK-J fragments to the binding of HIV-1 gp120. The variabledomains of VRC01 are shown in cartoon diagram and the gp120 bindingareas are masked with a surface.

FIG. 43 is the alignment of VRC01 Vh (SEQ ID NO: 1) to the ten closestgermline genes. Results were obtained from IgBLAST using the VRC01 Vhnucleotide sequence. Residue identities are shown as dots. Thenucleotide identity fraction for each of the ten germline genes, asreported by IgBLAST, is also shown.

FIG. 44 is the alignment of VRC01 Vk (SEQ ID NO: 2) to the ten closestgermline genes. Results were obtained from IgBLAST using the VRC01 Vknucleotide sequence up to amino acid residue Q90; since IGKV3-NL1*01 is‘not localized’, IGKV3-11*01 was selected as a top match for the Vkgermline. When comparing to IGKV3-11*01 using nucleotide sequences, thetwo-residue deletion in VRC01 Vk aligns to two S residues and involves aneighboring S->Y mutation. Residue identities are shown as dots. Thenucleotide identity fraction for each of the ten germline genes, asreported by IgBLAST, is also shown.

FIGS. 45A-45C is a set of graphs showing the results of a sequencecomparison of VRC01, VRC02 and VRC03 to a collection of gp140-bindingantibodies. FIG. 45A is a VH and Vk repertoire analysis for a collectionof gp140-binding antibodies and VRC01, 02 and 03. The pie charts displaythe distribution of V gene usage among the collection of uniqueantibodies. VRC01, 02, 03 genes are indicated with red arrows.

FIG. 45B shows a CDR amino acid length of the collection of antibodiescompared to VRC01, 02 and 03 indicated with a red arrow. CDR3 lengthswere determined according to NCBI IgBlast nomenclature with the CDR3region starting after the CTR and CVR amino acids for VRC01, 02 and 03,respectively. FIG. 45C shows the number of nucleotide mutations in the Vgenes of the antibody collection compared to VRC01, 02 and 03.

FIG. 46 is a set of graphs showing the correlations between the numberof affinity matured residues in VH and Vk and SPR determineddissociation constants, ELISA (EC50) binding, and neutralization (IC50)data for a set of VRC01 variants. VRC01 variants were made to revertinterface residues to their corresponding VH- and Vkgermline- encodedresidues in a series of single-, 4-, 7- or 12-residue mutations.

FIG. 47 is Table S1.

FIG. 48 is Table S2.

FIG. 49 is Table S3.

FIG. 50 is Table S4.

FIG. 51 is Table S5.

FIG. 52 is Table S6.

FIG. 53 is Table S7.

FIG. 54 is Table S8.

FIG. 55 is Table S9.

FIG. 56 is Table S10a.

FIG. 57 is Table S10b.

FIG. 58 is Table S11.

FIG. 59 is Table S12.

FIG. 60 is Table S13.

FIG. 61 is Table S14.

FIGS. 62A-62C is a set of electronic images a sequence alignment and abar graph showing F5 CDR H3 loop mutagenesis (SEQ ID NOs: 47-58). FIG.62A illustrates the structure of 2F5 Fab in complex with a gp41 peptide.The 2F5 CDR H3 contacts gp41 only at its base, while the tip extendsaway from the peptide. FIG. 62B is a close-up view of the 2F5 CDR H3loop. FIG. 62C shows mutations introduced into the tip of the 2F5 CDR H3(100A to 100F) along with a plot of the Wimley-White predicted freeenergies of transfer to a lipid bilayer interface or to octanol for eachof the mutations.

FIG. 63 is Table 1.

FIG. 64 is a set of graphs showing the neutralization of HxB2 by CDR H3mutants of antibody 2F5. Top, neutralization profiles of 2F5 variantswith single mutations to serine. Single serine substitutions resulted ina 15- to 500-fold reduction in neutralization potency. Middle,neutralization profiles of double mutations to serine. Double serinesubstitutions completely abrogated 2F5-mediated neutralization. Bottom,neutralization profiles of 2F5 variants with mutations to tryptophan.Tryptophan substitutions were either commensurate with or more potentthan wild-type 2F5. Neutralization curves for wild-type 2F5 IgG arecolored black and are shown in all three panels. 1D4, mouse antirhodopsin antibody used as a negative control.

FIGS. 65A-65C are a set of graphs showing the relationship between2F5-mediated HIV-1 neutralization and the hydrophobicity of its CDR H3loop. FIG. 65A shows 2F5 variant neutralization IC50s plotted againstthe calculated ΔG_(wif) of the 2F5 CDR H3 loop for each virus straintested. Linear regressions were fit to each individual group, and nosignificant differences were observed in the slopes of each of thecurves. The resulting P values of the correlations were statisticallysignificant (P<0.02) for all strains tested, with the exception ofRHPA-4259. FIG. 65B shows 2F5 variant neutralization IC50s plottedagainst affinity to gp41MPER peptide, with linear fits applied to eachgroup. No statistically significant associations were observed in thiscase, and the regressions appeared largely driven by the KD of 2F5variant HOOFS to gp41, likely because of minor contacts made by thisresidue with gp41. FIG. 65 C shows plots of relative free energies ofneutralization (ΔG^(N)) versus calculated ΔG_(wif) values, along withindividual solid lines) and shared (dashed black line and displayedstatistics) linear fits for all strains tested. HIV strains arerepresented as symbols and lines. The dashed vertical lines define theΔG_(wif) value of the wild-type 2F5 CDR H3 tip or the KD of wild-type2F5 to gp41 peptide.

FIG. 66 is Table 2.

FIGS. 67A-67C are a depiction of the threshold for effects of increased2F5 CDR H3 loop hydrophobicity. To account for the observed leveling offof the effects of increased 2F5 CDR H3 loop hydrophobicity, quadraticfits were applied to the data shown in FIG. 66. FIG. 67A, 2F5 variantneutralization IC50s plotted against the calculated ΔG_(wif) of the 2F5CDR H3 loop for each virus strain tested. Individual curves were fit toeach group using a quadratic regression model, which revealed nosignificant differences in their slopes or quadratic terms. FIG. 67B,plot of relative free energies of neutralization (ΔΔG^(N)) versuscalculated ΔG_(wif). As in panel A, individual curves were fit to eachgroup using a quadratic regression model, which revealed no significantdifferences in their slopes or quadratic terms. FIG. 67C, the quadraticmodels were refit to the data using common estimates of the slopes andquadratic terms and plotted as a shared quadratic regression for allstrains tested (dashed black line). A minimum of the ΔΔG^(N) wasobserved to occur at a ΔG_(wif) of −4.08 kcal/mol (arrow), representingthe maximal effect on neutralization beyond which increasedhydrophobicity of the CDR H3 loop had detrimental effects on2F5-mediated neutralization. HIV strains are represented as symbols andlines. The dashed vertical lines (gray) define the ΔG_(wif) value of thewild-type 2F5 CDR H3 tip.

FIG. 68 is schematic representation of the two-component mechanism of2F5-mediated neutralization of HIV-1. The free energy of 2F5-mediatedneutralization of HIV-1, ΔG^(N) (diagonal), can be expressed as the sumof the free energy of its structure-specific recognition of its gp41MPERepitope (vertical component) combined with the free energy ofnonspecific interactions mediated by the hydrophobic tip of its CDR H3loop (horizontal component). Structural representations of eachcomponent are shown.

FIG. 69 is Table s1.

FIG. 70 is Table s2.

FIG. 71 is Table s3.

FIG. 72 is a set of graphs showing the results of surface plasmonresonance binding profiles of 2F5 variants to gp41 peptide. Shown arerepresentative biacore binding profiles of gp41-MPER-C9 peptide towild-type and mutant 2F5 IgG's (black), overlaid with fits of the data.In all cases, IgGs were directly coupled to CM5 chips, and the peptidewas used as analyte. Plotted analyte concentratations are 2-fold serialdilutions ranging from 31 nM to 0.49 nM, except for 2F5 variantL100AS-F100BS, which ranged from 15 nM to 0.49 nM, and variantsL100ASV100DS and F100BS-1100FS, which ranged from 125 nM to 0.49 nM. Inall cases, nanomolar binding affinity to gp41 peptide was maintained.

FIGS. 73A-73D is a set of graphs of the relationship between HIV-1neutralization and ΔG_(oct), hydrophobicity of 2F5 CDR H3 P<0.0001variants. FIG. 73A, 2F5-variant neutralization IC50s plotted againstcalculated ΔG_(oct) of the 2F5 CDR H3 loop, for each virus straintested. Linear regressions were fit to each individual group, and nosignificant differences were observed in their slopes. The resultingP-values of the correlations were all statistically significant(P<0.05), with the exception of RHPA-4259. FIG. 73B shows that quadraticregressions fit to the same data as in FIG. 73A, reveal no significantdifferences in the slopes or quadratic terms. Plots of relative freeenergies of neutralization (ΔΔG^(N)) versus calculated ΔG_(oct) were fitwith linear (FIG. 73C) and quadratic regression models (FIG. 73D), asshown. Shared regressions across all strains in FIG. 73C and FIG. 73Dare represented as dashed black lines. A dashed vertical line definesthe AG, properties of the wild-type 2F5 CDR H3 tip.

FIG. 74A is a schematic diagram and the sequence of VRC01 ImmunoAdhesinHL (SEQ ID NO: 66).

FIG. 74B is a schematic diagram and the sequence of VRC01 ImmunoAdhesinLH (SEQ ID NO: 66).

FIGS. 75A-75E is a series of schematic diagrams for VRC8551, VRC8552,VRC9709, VRC9710, VRC9711, VRC9712, VRC9713, VRC9714, VRC9715, VRC9716,VRC9717, VRC9712, VRC9713 and VRC9174.

FIG. 76 is a table and an electronic image of a protein gel showing theexpression of chimeras of VRC01 and VRC03.

FIG. 77 is a set of graphs showing exemplary neutralization data forclade A HIV viruses obtained from VRC01 and VRC03 chimeric antibodies.

FIG. 78 is a set of graphs showing exemplary neutralization data forclade B HIV viruses obtained from VRC01 and VRC03 chimeric antibodies.

FIG. 79 is a set of graphs showing exemplary neutralization data forclade C HIV viruses obtained from VRC01 and VRC03 chimeric antibodies.

FIG. 80 is a sequence alignment of the amino acid sequence of the heavychain of antibody 57203 (SEQ ID NO: 1322) identified by 454 sequencing,the germline sequence IGHV1-02*02 (SEQ ID NO: 41), VRC01 (SEQ ID NO: 1),VRC02 (SEQ ID NO: 3) and VRC03 (SEQ ID NO: 27).

FIGS. 81A-81G are a set of graphs of exemplary neutralization data ofthe 57203 heavy chain VRC01 light chain chimera demonstrating that the57203 is VRC01 like, in that it complements the VRC01 light chain.

FIG. 82 is a graph showing the distribution of the VRC01 and VRC03sequences set for as SEQ ID NOs: 760-1459.

FIGS. 83-91 and 93-97 are supporting data showing the conserved gp120epitope common to the binding site of VRC01 and VRC03-like antibodies.

FIG. 92 shows the preduction of VCRO1 multimers.

FIGS. 97-117 are supporting data showing bioinformatics analysis.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the heavy chain ofgp120-specific antibody VRC01.

SEQ ID NO: 2 is the amino acid sequence of the light chain ofgp120-specific antibody VRC01.

SEQ ID NO: 3 is the amino acid sequence of the heavy chain ofgp120-specific antibody VRC02.

SEQ ID NO: 4 is the amino acid sequence of the light chain ofgp120-specific antibody VRC02.

SEQ ID NO: 5 is the amino acid sequence of the heavy chain ofgp41-specific antibody 2F5.

SEQ ID NO: 6 is the amino acid sequence of the heavy chain ofgp41-specific antibody 2F5 L_(100A)W.

SEQ ID NO: 7 is the amino acid sequence of the heavy chain ofgp41-specific antibody 2F5 F_(100B)W.

SEQ ID NO: 8 is the amino acid sequence of the heavy chain ofgp41-specific antibody 2F5 V_(100D)W.

SEQ ID NO: 9 is the amino acid sequence of the heavy chain ofgp41-specific antibody 2F5 L_(100A)W-V_(100D)W.

SEQ ID NO: 10 is the amino acid sequence of the light chain ofgp41-specific antibody 2F5.

SEQ ID NO: 11 is the gp41MPER Epitope Scaffold Immunological Probe ES1(1LGYA) Clade B.

SEQ ID NO: 12 is the gp41MPER Epitope Scaffold Immunological Probe ES1(1LGYA) Clade B point mutant.

SEQ ID NO: 13 is the gp41MPER Epitope Scaffold Immunological Probe ES1(1LGYA) Clade C.

SEQ ID NO: 14 is the gp41MPER Epitope Scaffold Immunological Probe ES2(1KU2A-s) Clade B.

SEQ ID NO: 15 is the gp41MPER Epitope Scaffold Immunological Probe ES2(1KU2A-s) Clade B point mutant.

SEQ ID NO: 16 is the gp41MPER Epitope Scaffold Immunological Probe ES2(1KU2A-s) Clade C.

SEQ ID NO: 17 is the gp41MPER Epitope Scaffold Immunological Probe ES3(2MATA) Clade B.

SEQ ID NO: 18 is the gp41MPER Epitope Scaffold Immunological Probe ES3(2MATA) Clade B point mutant.

SEQ ID NO: 19 is the gp41MPER Epitope Scaffold Immunological Probe ES3(2MATA) Clade C.

SEQ ID NO: 20 is the gp41MPER Epitope Scaffold Immunological Probe ES4(11WLA) Clade B.

SEQ ID NO: 21 is the gp41MPER Epitope Scaffold Immunological Probe ES4(11WLA) Clade B point mutant.

SEQ ID NO: 22 is the gp41MPER Epitope Scaffold Immunological Probe ES4(11WLA) Clade C.

SEQ ID NO: 23 is the gp41MPER Epitope Scaffold Immunological Probe ES5(1D3BB) Clade B.

SEQ ID NO: 24 is the gp41MPER Epitope Scaffold Immunological Probe ES5(1D3BB) Clade B point mutant.

SEQ ID NO: 25 is the gp41MPER Epitope Scaffold Immunological Probe ES5(1D3BB) Clade C.

SEQ ID NO: 26 is the Avitag amino acid sequence.

SEQ ID NO: 27 is the amino acid sequence of the heavy chain ofgp120-specific antibody VRC03.

SEQ ID NO: 28 is the amino acid sequence of the light chain ofgp120-specific antibody VRC03.

SEQ ID NO: 29 is the nucleic acid sequence of the heavy chain ofgp120-specific antibody VRC01.

SEQ ID NO: 30 is the nucleic acid sequence of the light chain ofgp120-specific antibody VRC01.

SEQ ID NO: 31 is the nucleic acid sequence of the heavy chain ofgp120-specific antibody VRC02.

SEQ ID NO: 32 is the nucleic acid sequence of the light chain ofgp120-specific antibody VRC02.

SEQ ID NO: 33 is the nucleic acid sequence of the heavy chain ofgp120-specific antibody VRC03.

SEQ ID NO: 34 is the nucleic acid sequence of the light chain ofgp120-specific antibody VRC03.

SEQ ID NO: 35 is the nucleic acid sequence of VRC01 ImmunoAdhesin HL.

SEQ ID NO: 36 is the amino acid sequence of an exemplary mouseinterleukin-2 (IL-2) leader sequence.

SEQ ID NO: 37 is the amino acid sequence of an exemplary gp120 V1/V2loop.

SEQ ID NO: 38 is the amino acid sequence of an exemplary gp120 V3 loop.

SEQ ID NO: 39 is the amino acid sequence of an exemplary peptide linker

SEQ ID NO: 40 is the amino acid sequence of an en exemplary peptidecorresponding to residues 657 to 669 of linked to a C-terminal C9 tag.

SEQ ID NO: 41 is the amino acid sequence of the heavy chain ofIGHV1-02*02.

SEQ ID NO: 42 is the amino acid sequence of the VK gene IGKV3-11*01.

SEQ ID NO: 43 is the amino acid sequence of the VK gene IGKV3-20*01.

SEQ ID NO: 44 is the amino acid sequence for the wild type clade B HXBc2core gp120.

SEQ ID NO: 45 is the amino acid sequence for the wild type clade A/E93TH057 core gp120.

SEQ ID NO: 46 is the amino acid sequence of aV5 region of a gp120.

SEQ ID NOs: 47-58 are the amino acids sequences of 2F5 CDR H3 loopmutants.

SEQ ID NO: 59 is the amino acid sequence of a V1/V2 trim sequence ofgp120 from HIV SEQ ID NO: 60 is the nucleic acid sequence of VRC01ImmunoAdhesin LH.

SEQ ID NOs: 61-759 are nucleic acid sequence of the heavy chains ofVRC01 and VRC03-like antibodies.

SEQ ID NOs: 760-1459 are amino acid sequence of the heavy chains ofVRC01 and VRC03-like antibodies.

SEQ ID NOs: 1460-1505 are germline sequences for VRC01-like antibodies.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence.txt, which was created on Mar. 22, 2012, and is˜1.3 MB bytes, which is incorporated by reference herein.

DETAILED DESCRIPTION I. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). Terms describing proteinstructure and structural elements of proteins can be found in Creighton,Proteins, Structures and Molecular Properties, W.H. Freeman & Co., NewYork, 1993 (ISBN 0-717-7030) which is incorporated by reference hereinin its entirety.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of this disclosure, thefollowing explanations of terms are provided:

Administration:

The introduction of a composition into a subject by a chosen route.Administration can be local or systemic. For example, if the chosenroute is intravenous, the composition is administered by introducing thecomposition into a vein of the subject. In some examples a disclosedantibody specific for an HIV protein or polypeptide is administered to asubject.

Amino Acid Substitution:

The replacement of one amino acid in peptide with a different aminoacid.

Amplification:

A technique that increases the number of copies of a nucleic acidmolecule (such as an RNA or DNA). An example of amplification is thepolymerase chain reaction, in which a biological sample is contactedwith a pair of oligonucleotide primers, under conditions that allow forthe hybridization of the primers to a nucleic acid template in thesample. The primers are extended under suitable conditions, dissociatedfrom the template, and then re-annealed, extended, and dissociated toamplify the number of copies of the nucleic acid. The product ofamplification can be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing using standard techniques.Other examples of amplification include strand displacementamplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed in EP-A-320308; gap filling ligase chain reaction amplification, as disclosed inU.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134.

Animal:

Living multi-cellular vertebrate organisms, a category that includes,for example, mammals and birds. The term mammal includes both human andnon-human mammals. Similarly, the term “subject” includes both human andveterinary subjects.

Antibody:

A polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or antigen binding fragments thereof, whichspecifically binds and recognizes an analyte (antigen) such as gp120, orgp41 or an antigenic fragment of gp120 or gp41. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable regiongenes.

Antibodies exist, for example as intact immunoglobulins and as a numberof well characterized fragments produced by digestion with variouspeptidases. For instance, Fabs, Fvs, and single-chain Fvs (scFvs) thatspecifically bind to gp120 or fragments of gp120 would be gp120-specificbinding agents. Similarly, Fabs, Fvs, scFvs that specifically bind togp41 or fragments of gp41 would be gp41-specific binding agents. A scFvprotein is a fusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term also includes genetically engineered forms such as chimericantibodies (such as humanized murine antibodies), heteroconjugateantibodies such as bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Antibody fragments are defined as follows: (1) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody moleculeproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (3) (Fab′)₂, the fragment of the antibody obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference in its entirety). Thus one of ordinary skill in the art willrecognize the numbering of the residues in the disclosed antibodies ismade with reference to the Kabat convention. The Kabat database is nowmaintained online. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. Light chain CDRs are sometimes referred to as CDRL1, CDR L2, and CDR L3. Heavy chain CDRs are sometimes referred to asCDR H1, CDR H2, and CDR H3.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody fragment, suchas Fv, scFv, dsFv or Fab. References to “V_(L)” or “VL” refer to thevariable region of an immunoglobulin light chain, including that of anFv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies. In some examples monoclonal antibodies are isolated from asubject. The amino acid sequences of such isolated monoclonal antibodiescan be determined.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, such as at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (for example, see U.S. Pat.No. 5,585,089).

Antigen:

A compound, composition, or substance that can stimulate the productionof antibodies or a T cell response in an animal, including compositionsthat are injected or absorbed into an animal. An antigen reacts with theproducts of specific humoral or cellular immunity, including thoseinduced by heterologous antigens, such as the disclosed antigens.“Epitope” or “antigenic determinant” refers to the region of an antigento which B and/or T cells respond. In one embodiment, T cells respond tothe epitope, when the epitope is presented in conjunction with an MHCmolecule. Epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5,about 9, or about 8-10 amino acids in a unique spatial conformation.Methods of determining spatial conformation of epitopes include, forexample, x-ray crystallography and nuclear magnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. In some examples, antigensinclude peptides derived from a pathogen of interest. Exemplarypathogens include bacteria, fungi, viruses and parasites. In specificexamples, an antigen is derived from HIV, such as a gp120 polypeptide orantigenic fragment thereof, such as a gp120 outer domain or fragmentthereof. In other examples an antigen a gp41 polypeptide derived fromHIV or antigenic fragment thereof.

A “target epitope” is a specific epitope on an antigen that specificallybinds a antibody of interest, such as a monoclonal antibody. In someexamples, a target epitope includes the amino acid residues that contactthe antibody of interest, such that the target epitope can be selectedby the amino acid residues determined to be in contact with the antibodyof interest.

Antigenic Surface:

A surface of a molecule, for example a protein such as a gp120 proteinor polypeptide, capable of eliciting an immune response. An antigenicsurface includes the defining features of that surface, for example thethree-dimensional shape and the surface charge. An antigenic surfaceincludes both surfaces that occur on gp120 polypeptides as well assurfaces of compounds that mimic the surface of a gp120 polypeptide(mimetics). In some examples, an antigenic surface include all or partof the surface of gp120 that binds to the CD4 receptor.

Atomic Coordinates or Structure Coordinates:

Mathematical coordinates derived from mathematical equations related tothe patterns obtained on diffraction of a monochromatic beam of X-raysby the atoms (scattering centers) such as an antigen, or an antigen incomplex with an antibody. In some examples that antigen can be gp120, agp120:antibody complex, or combinations thereof in a crystal. In someexamples that antigen can be gp41, a gp41:antibody complex, orcombinations thereof in a crystal. The diffraction data are used tocalculate an electron density map of the repeating unit of the crystal.The electron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal. In one example,the term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays, such as by the atoms of agp120 in crystal form.

Those of ordinary skill in the art understand that a set of structurecoordinates determined by X-ray crystallography is not without standarderror. For the purpose of this disclosure, any set of structurecoordinates that have a root mean square deviation of protein backboneatoms (N, Cα, C and 0) of less than about 1.0 Angstroms whensuperimposed, such as about 0.75, or about 0.5, or about 0.25 Angstroms,using backbone atoms, shall (in the absence of an explicit statement tothe contrary) be considered identical.

Binding Affinity

Affinity of an antibody or antigen binding fragment thereof for anantigen. In one embodiment, affinity is calculated by a modification ofthe Scatchard method described by Frankel et al., Mol. Immunol.,16:101-106, 1979. In another embodiment, binding affinity is measured byan antigen/antibody dissociation rate. In yet another embodiment, a highbinding affinity is measured by a competition radioimmunoassay. Inseveral examples, a high binding affinity is at least about 1×10⁻⁸ M. Inother embodiments, a high binding affinity is at least about 1.5×10⁻⁸,at least about 2.0×10⁻⁸, at least about 2.5×10⁻⁸, at least about3.0×10⁻⁸, at least about 3.5×10⁻⁸, at least about 4.0×10⁻⁸, at leastabout 4.5×10⁻⁸, or at least about 5.0×10⁻⁸ M.

CD4:

Cluster of differentiation factor 4 polypeptide; a T-cell surfaceprotein that mediates interaction with the MHC class II molecule. CD4also serves as the primary receptor site for HIV on T-cells during HIV-Iinfection. CD4 is known to bind to gp120 from HIV. The known sequence ofthe CD4 precursor has a hydrophobic signal peptide, an extracellularregion of approximately 370 amino acids, a highly hydrophobic stretchwith significant identity to the membrane-spanning domain of the classII MHC beta chain, and a highly charged intracellular sequence of 40resides (Maddon, Cell 42:93, 1985).

The term “CD4” includes polypeptide molecules that are derived from CD4include fragments of CD4, generated either by chemical (for exampleenzymatic) digestion or genetic engineering means. Such a fragment maybe one or more entire CD4 protein domains. The extracellular domain ofCD4 consists of four contiguous immunoglobulin-like regions (D1, D2, D3,and D4, see Sakihama et al., Proc. Natl. Acad. Sci. 92:6444, 1995; U.S.Pat. No. 6,117,655), and amino acids 1 to 183 have been shown to beinvolved in gp120 binding. For instance, a binding molecule or bindingdomain derived from CD4 would comprise a sufficient portion of the CD4protein to mediate specific and functional interaction between thebinding fragment and a native or viral binding site of CD4. One suchbinding fragment includes both the D1 and D2 extracellular domains ofCD4 (D1D2 is also a fragment of soluble CD4 or sCD4 which is comprisedof D1 D2 D3 and D4), although smaller fragments may also providespecific and functional CD4-like binding. The gp120-binding site hasbeen mapped to D1 of CD4.

CD4 polypeptides also include “CD4-derived molecules” which encompassesanalogs (non-protein organic molecules), derivatives (chemicallyfunctionalized protein molecules obtained starting with the disclosedprotein sequences) or mimetics (three-dimensionally similar chemicals)of the native CD4 structure, as well as proteins sequence variants orgenetic alleles that maintain the ability to functionally bind to atarget molecule.

CD4BS Antibodies:

Antibodies that bind to or substantially overlap the CD4 binding surfaceof a gp120 polypeptide. The antibodies interfere with or prevent CD4from binding to a gp120 polypeptide.

CD4i Antibodies:

Antibodies that bind to a conformation of gp120 induced by CD4 binding.

Chimeric Antibody:

An antibody which includes sequences derived from two differentantibodies, which typically are of different species. In some examples,a chimeric antibody includes one or more CDRs and/or framework regionsfrom one human antibody and CDRs and/or framework regions from anotherhuman antibody.

Contacting:

Placement in direct physical association; includes both in solid andliquid form, which can take place either in vivo or in vitro. Contactingincludes contact between one molecule and another molecule, for examplethe amino acid on the surface of one polypeptide, such as an antigen,that contacts another polypeptide, such as an antibody. Contacting canalso include contacting a cell for example by placing an antibody indirect physical association with a cell.

Computer Readable Media:

Any medium or media, which can be read and accessed directly by acomputer, so that the media is suitable for use in a computer system.Such media include, but are not limited to: magnetic storage media suchas floppy discs, hard disc storage medium and magnetic tape; opticalstorage media such as optical discs or CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media.

Computer System:

Hardware that can be used to analyze atomic coordinate data and/ordesign an antigen using atomic coordinate data. The minimum hardware ofa computer-based system typically comprises a central processing unit(CPU), an input device, for example a mouse, keyboard, and the like, anoutput device, and a data storage device. Desirably a monitor isprovided to visualize structure data. The data storage device may be RAMor other means for accessing computer readable. Examples of such systemsare microcomputer workstations available from Silicon GraphicsIncorporated and Sun Microsystems running Unix based Windows NT or IBMOS/2 operating systems.

Epitope:

An antigenic determinant. These are particular chemical groups orpeptide sequences on a molecule that are antigenic, i.e. that elicit aspecific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. In some examples a disclosedantibody specifically binds to an epitope on the surface of gp120 fromHIV. In some examples a disclosed antibody specifically binds to anepitope on the surface of gp41 from HIV.

Epitope Scaffold

Refers to a heterologous protein that is engrafted with a foreignepitope of interest on its surface. Transplantation of the epitope isperformed computationally in a manner that preserves its relevantstructure and conformation. Mutations within the acceptor scaffold aremade in order to accommodate the epitope graft. The graft can bemodified to represent the sequences of different clades and strains.

Framework Region

Amino acid sequences interposed between CDRs. Includes variable lightand variable heavy framework regions. The framework regions serve tohold the CDRs in an appropriate orientation for antigen binding.

Fc Polypeptide

The polypeptide comprising the constant region of an antibody excludingthe first constant region immunoglobulin domain. Fc region generallyrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, and the last three constant region immunoglobulin domainsof IgE and IgM. An Fc region may also include part or all of theflexible hinge N-terminal to these domains. For IgA and IgM, an Fcregion may or may not comprise the tailpiece, and may or may not bebound by the J chain. For IgG, the Fc region comprises immunoglobulindomains Cgamma2 and Cgamma3 (Cγ₂ and Cγ₃) and the lower part of thehinge between Cgamma1 (Cγ1) and Cγ₂. Although the boundaries of the Fcregion may vary, the human IgG heavy chain Fc region is usually definedto comprise residues C226 or P230 to its carboxyl-terminus, wherein thenumbering is according to the EU index as in Kabat. For IgA, the Fcregion comprises immunoglobulin domains Calpha2 and Calpha3 (Cα2 andCα3) and the lower part of the hinge between Calpha 1 (Cα1) and Cα2.Encompassed within the definition of the Fc region are functionallyequivalent analogs and variants of the Fc region. A functionallyequivalent analog of the Fc region may be a variant Fc region,comprising one or more amino acid modifications relative to thewild-type or naturally existing Fc region. Variant Fc regions willpossess at least 50% homology with a naturally existing Fc region, suchas about 80%, and about 90%, or at least about 95% homology.Functionally equivalent analogs of the Fc region may comprise one ormore amino acid residues added to or deleted from the N- or C-termini ofthe protein, such as no more than 30 or no more than 10 additions and/ordeletions. Functionally equivalent analogs of the Fc region include Fcregions operably linked to a fusion partner. Functionally equivalentanalogs of the Fc region must comprise the majority of all of the Igdomains that compose Fc region as defined above; for example IgG and IgAFc regions as defined herein must comprise the majority of the sequenceencoding CH₂ and the majority of the sequence encoding CH₃. Thus, theCH₂ domain on its own, or the CH₃ domain on its own, are not consideredFc region. The Fc region may refer to this region in isolation, or thisregion in the context of an Fc fusion polypeptide (immunoadhesin, seebelow).

Furin

A calcium dependent serine endoprotease that cleaves precursor proteinsat paired basic amino acid processing sites. In vivo, substrates offurin include proparathyroid hormone, proablumin, and von Willebrandfactgor. Furin can also cleave HIV envelope protein gp160 into gp120 andgp41. Furin cleaves proteins just downstream of a basic amino acidtarget sequence (canonically, Arg-X-(Arg/Lys)-Arg'). Thus, this aminoacid sequence is a furin cleavage site.

gp41:

A HIV protein that contains a transmembrane domain and remains in atrimeric configuration; it interacts with gp120 in a non-covalentmanner. The envelope protein of HIV-1 is initially synthesized as alonger precursor protein of 845-870 amino acids in size, designatedgp160. gp160 forms a homotrimer and undergoes glycosylation within theGolgi apparatus. In vivo, it is then cleaved by a cellular protease intogp120 and gp41. The amino acid sequence of an example of gp41 is setforth in GENBANK® Accession No. CAD20975 (as available on Oct. 16, 2009)which is incorporated by reference herein. It is understood that thesequence of gp41 can vary from that given in GENBANK® Accession No.CAD20975. gp41 contains a transmembrane domain and typically remains ina trimeric configuration; it interacts with gp120 in a non-covalentmanner.

gp120:

An envelope protein from Human Immunodeficiency Virus (HIV). Thisenvelope protein is initially synthesized as a longer precursor proteinof 845-870 amino acids in size, designated gp160. gp160 is cleaved by acellular protease into gp120 and gp41. gp120 contains most of theexternal, surface-exposed, domains of the HIV envelope glycoproteincomplex, and it is gp120 which binds both to cellular CD4 receptors andto cellular chemokine receptors (such as CCR5).

The mature gp120 wildtype polypeptides have about 500 amino acids in theprimary sequence. gp120 is heavily N-glycosylated giving rise to anapparent molecular weight of 120 kD. The polypeptide is comprised offive conserved regions (C1-C5) and five regions of high variability(V1-V5). Exemplary sequence of wt gp120 polypeptides are shown onGENBANK®, for example accession numbers AAB05604 and AAD12142 (asavailable on Oct. 16, 2009), incorporated by reference herein. It isunderstood that there are numerous variation in the sequence of gp120from what is given in GENBANK®, for example accession numbers AAB05604and AAD12142, and that these variants are skill recognized in the art asgp120.

The gp120 core has a molecular structure, which includes two domains: an“inner” domain (which faces gp41) and an “outer” domain (which is mostlyexposed on the surface of the oligomeric envelope glycoprotein complex).The two gp120 domains are separated by a “bridging sheet” that is notpart of either domain. The gp120 core comprises 25 beta strands, 5 alphahelices, and 10 defined loop segments.

The third variable region referred to herein as the V3 loop is a loop ofabout 35 amino acids critical for the binding of the co-receptor anddetermination of which of the co-receptors will bind. In certainexamples the V3 loop comprises residues 296-331.

The numbering used in gp120 polypeptides disclosed herein is relative tothe HXB2 numbering scheme as set forth in Numbering Positions in HIVRelative to HXB2CG Bette Korber et al., Human Retroviruses and AIDS1998:A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences.Korber B, Kuiken C L, Foley B, Hahn B, McCutchan F, Mellors J W, andSodroski J, Eds. Theoretical Biology and Biophysics Group, Los AlamosNational Laboratory, Los Alamos, N M which is incorporated by referenceherein in its entirety.

Host Cells:

Cells in which a vector can be propagated and its DNA expressed, forexample a disclosed antibody can be expressed in a host cell. The cellmay be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Immunoadhesin:

A molecular fusion of a protein with the Fc region of an immunoglobulin,wherein the immunogloblin retains specific properties, such as Fcreceptor binding and increased half-life. An Fc fusion combines the Fcregion of an immunoglobulin with a fusion partner, which in general canbe any protein, polypeptide, peptide, or small molecule. In one example,and immunoadhesin includes the hinge, CH₂, and CH₃ domains of theimmunoglobulin gamma 1 heavy chain constant region. In another example,the immunoadhesin includes the CH₂, and CH₃ domains of an IgG.

Immunological Probe:

A molecule that can be used for selection of antibodies from sera whichare directed against a specific epitope, including from human patientsera. The epitope scaffolds, along with related point mutants, can beused as immunological probes in both positive and negative selection ofantibodies against the epitope graft. In some examples immunologicalprobes are engineered variants of gp120.

Immunologically Reactive Conditions:

Includes reference to conditions which allow an antibody raised againsta particular epitope to bind to that epitope to a detectably greaterdegree than, and/or to the substantial exclusion of, binding tosubstantially all other epitopes. Immunologically reactive conditionsare dependent upon the format of the antibody binding reaction andtypically are those utilized in immunoassay protocols or thoseconditions encountered in vivo. See Harlow & Lane, supra, for adescription of immunoassay formats and conditions. The immunologicallyreactive conditions employed in the methods are “physiologicalconditions” which include reference to conditions (e.g., temperature,osmolarity, pH) that are typical inside a living mammal or a mammaliancell. While it is recognized that some organs are subject to extremeconditions, the intra-organismal and intracellular environment normallylies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to7.5), contains water as the predominant solvent, and exists at atemperature above 0° C. and below 50° C. Osmolarity is within the rangethat is supportive of cell viability and proliferation.

IgA:

A polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin alpha gene. Inhumans, this class or isotype comprises IgA₁ and IgA₂. IgA antibodiescan exist as monomers, polymers (referred to as pIgA) of predominantlydimeric form, and secretory IgA. The constant chain of wild-type IgAcontains an 18-amino-acid extension at its C-terminus called the tailpiece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDapeptide called the J chain linking two monomers of IgA through theconserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotype of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans, this class comprises IgG₁, IgG₂, IgG₃, and IgG₄. In mice, thisclass comprises IgG₁, IgG_(2a), IgG_(2b), IgG_(3.)

Inhibiting or Treating a Disease:

Inhibiting the full development of a disease or condition, for example,in a subject who is at risk for a disease such as acquiredimmunodeficiency syndrome (AIDS). “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

Isolated:

An “isolated” biological component (such as a cell, for example a Bcell,a nucleic acid, peptide, protein or antibody) has been substantiallyseparated, produced apart from, or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, such as, other chromosomal and extrachromosomal DNA and RNA, andproteins. Nucleic acids, peptides and proteins which have been“isolated” thus include nucleic acids and proteins purified by standardpurification methods. The term also embraces nucleic acids, peptides,and proteins prepared by recombinant expression in a host cell as wellas chemically synthesized nucleic acids. In some examples an antibody,such as an antibody specific for gp120 can be isolated, for exampleisolated from a subject infected with HIV.

In Silico

A process performed virtually within a computer. For example, using acomputer, a virtual compound can be screened for surface similarity orconversely surface complementarity to a virtual representation of theatomic positions at least a portion of a gp120 polypeptide, a gp120polypeptide in complex with an antibody, a gp41 polypeptide, or a pg41polypeptide in complex with an antibody.

K_(d):

The dissociation constant for a given interaction, such as a polypeptideligand interaction or an antibody antigen interaction. For example, forthe bimolecular interaction of an antibody (such as VRC01, VRC02, orVRC03) and an antigen (such as gp120) it is the concentration of theindividual components of the bimolecular interaction divided by theconcentration of the complex.

Label:

A detectable compound or composition that is conjugated directly orindirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In some examples, a disclosed antibody as labeled.

Neutralizing Antibody:

An antibody which reduces the infectious titer of an infectious agent bybinding to a specific antigen on the infectious agent. In some examplesthe infectious agent is a virus. In some examples, an antibody that isspecific for gp120 neutralizes the infectious titer of HIV.

Nucleic Acid:

A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, e.g., a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (e.g., promoter, origin of replication,ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology (Ausubelet al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

A polynucleotide or nucleic acid sequence refers to a polymeric form ofnucleotide at least 10 bases in length. A recombinant polynucleotideincludes a polynucleotide that is not immediately contiguous with bothof the coding sequences with which it is immediately contiguous (one onthe 5′ end and one on the 3′ end) in the naturally occurring genome ofthe organism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms of DNA.A gp120 polynucleotide is a nucleic acid encoding a gp120 polypeptide.

Pharmaceutically Acceptable Carriers:

The pharmaceutically acceptable carriers of use are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition, 1995, describes compositions andformulations suitable for pharmaceutical delivery of the antibodiesherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical Agent:

A chemical compound or composition capable of inducing a desiredtherapeutic or prophylactic effect when properly administered to asubject or a cell. In some examples a pharmaceutical agent includes oneone or more of the disclosed antibodies.

Polypeptide:

Any chain of amino acids, regardless of length or post-translationalmodification (e.g., glycosylation or phosphorylation). In oneembodiment, the polypeptide is gp120 polypeptide. In another embodiment,the polypeptide is gp41 polypeptide. In one embodiment, the polypeptideis a disclosed antibody or a fragment thereof. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal end.

Purified:

The term purified does not require absolute purity; rather, it isintended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein (such as an antibody)is more enriched than the peptide or protein is in its naturalenvironment within a cell. In one embodiment, a preparation is purifiedsuch that the protein or peptide represents at least 50% of the totalpeptide or protein content of the preparation.

Recombinant:

A recombinant nucleic acid is one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques.

Sequence Identity:

The similarity between amino acid sequences is expressed in terms of thesimilarity between the sequences, otherwise referred to as sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. Homologs or variantsof a polypeptide will possess a relatively high degree of sequenceidentity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Specifically Bind:

When referring to an antibody, refers to a binding reaction whichdetermines the presence of a target protein, peptide, or polysaccharidein the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated conditions, an antibody bindspreferentially to a particular target protein, peptide or polysaccharide(such as an antigen present on the surface of a pathogen, for examplegp120) and do not bind in a significant amount to other proteins orpolysaccharides present in the sample or subject. Specific binding canbe determined by methods known in the art. With reference to an antibodyantigen complex, specific binding of the antigen and antibody has aK_(d) of less than about 10⁻⁶ Molar, such as less than about 10⁻⁶ Molar,10⁻⁷ Molar, 10⁻⁸ Molar, 10⁻⁹, or even less than about 10⁻¹⁹ Molar.

Therapeutic Agent:

Used in a generic sense, it includes treating agents, prophylacticagents, and replacement agents.

Therapeutically Effective Amount:

A quantity of a specific substance, such as a disclosed antibody,sufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit HIV replication ortreat AIDS. In several embodiments, a therapeutically effective amountis the amount necessary to reduce a sign or symptom of AIDS, and/or todecrease viral titer in a subject. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations that has been shown to achieve a desired in vitro effect.

T Cell:

A white blood cell critical to the immune response. T cells include, butare not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyteis an immune cell that carries a marker on its surface known as “clusterof differentiation 4” (CD4). These cells, also known as helper T cells,help orchestrate the immune response, including antibody responses aswell as killer T cell responses. CD8⁺ T cells carry the “cluster ofdifferentiation 8” (CD8) marker. In one embodiment, a CD8 T cells is acytotoxic T lymphocytes. In another embodiment, a CD8 cell is asuppressor T cell.

Vector:

A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Virus:

Microscopic infectious organism that reproduces inside living cells. Avirus consists essentially of a core of a single nucleic acid surroundedby a protein coat, and has the ability to replicate only inside a livingcell. “Viral replication” is the production of additional virus by theoccurrence of at least one viral life cycle. A virus may subvert thehost cells' normal functions, causing the cell to behave in a mannerdetermined by the virus. For example, a viral infection may result in acell producing a cytokine, or responding to a cytokine, when theuninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-I and HIV-II), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV).

HIV-I is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome (AIDS). “HIV disease” refers to awell-recognized constellation of signs and symptoms (including thedevelopment of opportunistic infections) in persons who are infected byan HIV virus, as determined by antibody or western blot studies.Laboratory findings associated with this disease are a progressivedecline in T cells.

II. Description of Several Embodiments

A. Neutralizing Monoclonal Antibodies

Isolated human monoclonal antibodies that specifically bind gp120 orgp41 are disclosed herein. Also disclosed herein are compositionsincluding these human monoclonal antibodies and a pharmaceuticallyacceptable carrier. Nucleic acids encoding these antibodies, expressionvectors comprising these nucleic acids, and isolated host cells thatexpress the nucleic acids are also provided.

Compositions comprising the human monoclonal antibodies specific forgp120 or gp41 can be used for research, diagnostic and therapeuticpurposes. For example, the human monoclonal antibodies disclosed hereincan be used to diagnose or treat a subject having an HIV-1 infectionand/or AIDS. For example, the antibodies can be used to determine HIV-1titer in a subject. The antibodies disclosed herein also can be used tostudy the biology of the human immunodeficiency virus.

In some embodiments, the isolated human monoclonal antibody specificallybinds gp120, and includes a heavy chain with amino acids 26-33 (CDR1),51-58 (CDR2), and 97-110 (CDR3) of SEQ ID NO: 1. In specific examples,the heavy chain of the human monoclonal antibody includes SEQ ID NO: 1.In additional examples, the heavy chain of the monoclonal antibodyincludes SEQ ID NO: 3. In another example, the isolated human monoclonalantibody specifically binds gp120, and includes a heavy chain with aminoacids 26-33 (CDR1), 51-58 (CDR2), and 97-110 (CDR3) of SEQ ID NO: 1 andthe light chain of the antibody includes amino acids 27-30 (CDR1), 48-50(CDR2), and 87-91 (CDR3) of SEQ ID NO: 2. In additional embodiments, theisolated human monoclonal antibody specifically binds gp120 and thelight chain of the antibody includes amino acids 27-30 (CDR1), 48-50(CDR2), and 87-91 (CDR3) of SEQ ID NO: 2. In specific examples, thelight chain of the antibody includes SEQ ID NO: 2. In additionalexamples, the light chain of the antibody includes SEQ ID NO: 4.

In further embodiments, the isolated human monoclonal antibodyspecifically binds gp120, and includes a heavy chain with at most one,at most two, at most three or at most four amino acid substitutions inamino acids 26-33 (CDR1), 51-58 (CDR2), and 97-110 (CDR3) of SEQ ID NO:1, and a light chain. In some embodiments, the antibody can include aheavy chain with at most one, at most two, at most three or at most fouramino acid substitutions in amino acids in 26-33 (CDR1), 51-58 (CDR2),and 97-110 (CDR3) of SEQ ID NO: 1, and can include a light chain thatincludes amino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQID NO: 2. In some embodiments, the antibody can include a heavy chainwith at most one, at most two, at most three or at most four amino acidsubstitutions amino acids in 26-33 (CDR1), 51-58 (CDR2), and 97-110(CDR3) of SEQ ID NO: 1, and can include a light chain with at most one,at most two, at most three or at most four amino acid substitutions inamino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO:2.

The antibody can include a heavy chain with at most one, at most two, atmost three or at most four amino acid substitutions amino acids in 26-33(CDR1), 51-58 (CDR2), and 97-110 (CDR3) of SEQ ID NO: 1 and a lightchain that can include SEQ ID NO: 2 or SEQ ID NO: 4. In some examples,these antibodies retain the binding affinity of the parental antibody(VRC01 or VRC02) for the antigenic epitope. Thus, in some examples,these antibodies have a KD of <3 nM for the antigenic epitope of gp120.

In another set of embodiments, the isolated human monoclonal antibodyspecifically binds gp120, and the light chain of the antibody includesat most one, at most two, at most three or at most four substitutions inamino acids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO:2. It should be noted that no more than three substitutions can be madein CDR2. The isolated monoclonal antibody can include a heavy chain thatincludes amino acids 26-33 (CDR1), 51-58 (CDR2), and 97-110 (CDR3) ofSEQ ID NO: 1 and the light chain of the antibody can include at mostone, at most two, at most three or at most four substitutions in aminoacids 27-30 (CDR1), 48-50 (CDR2), and 87-91 (CDR3) of SEQ ID NO: 2. Itshould be noted that no more than three substitutions can be made inCDR2. The antibody can include a heavy chain including the amino acidsequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3, and a light chainthat includes SEQ ID NO: 2 with at most one, at most two, at most threeor at most four substitutions in amino acids 27-30 (CDR1), 48-50 (CDR2),and 87-91 (CDR3) of SEQ ID NO: 2. It should be noted that no more thanthree substitutions can be made in CDR2. In some examples, theseantibodies retain the binding affinity of the parental antibody (VRC01or VRC02) for the antigenic epitope. Thus, in some examples, theseantibodies have a K_(D) of <3 nM for the antigenic epitope of gp120.

In some embodiments, the isolated human monoclonal antibody specificallybinds gp120, and includes a heavy chain with amino acids 26-35 (CDR1),50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO: 27. In specific examples,the heavy chain of the human monoclonal antibody includes SEQ ID NO: 27.In another example, the isolated human monoclonal antibody specificallybinds gp120, and includes a heavy chain with amino acids 26-35 (CDR1),50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO: 27 and a light chain thatincludes amino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQID NO: 28.

In further embodiments, the isolated human monoclonal antibodyspecifically binds gp120, and includes a heavy chain with at most one,at most two, at most three or at most four amino acid substitutions inamino acids 26-35 (CDR1), 50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO:27, and a light chain. The antibody can include a heavy chain with atmost one, at most two, at most three or at most four amino acidsubstitutions amino acids in 26-35 (CDR1), 50-66 (CDR2), and 106-119(CDR3) of SEQ ID NO: 27, and a light chain that includes amino acids24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO: 28. Theantibody can include a heavy chain with at most one, at most two, atmost three or at most four amino acid substitutions amino acids in 26-35(CDR1), 50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO: 27 and the lightchain of the antibody can include SEQ ID NO: 28. In some examples, theseantibodies retain the binding affinity of the parental antibody (VRC03)for the antigenic epitope.

In another set of embodiments, the isolated human monoclonal antibodyspecifically binds gp120, and the light chain of the antibody includesat most one, at most two, at most three or at most four substitutions inamino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO:28. The isolated monoclonal antibody can include a heavy chain thatincludes 26-35 (CDR1), 50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO: 27and a light chain that include at most one, at most two, at most threeor at most four substitutions in amino acids 24-33 (CDR1), 49-55 (CDR2),and 88-92 (CDR3) of SEQ ID NO: 28. The antibody can include a heavychain including the amino acid sequence set forth as SEQ ID NO: 27, anda light chain that includes at most one, at most two, at most three orat most four substitutions in amino acids 24-33 (CDR1), 49-55 (CDR2),and 88-92 (CDR3) of SEQ ID NO: 28. In some examples, these antibodiesretain the binding affinity of the parental antibody (VRC03) for theantigenic epitope. In additional embodiments, the isolated humanmonoclonal antibody specifically binds gp120 and the light chain of theantibody includes amino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92(CDR3) of SEQ ID NO: 28. In specific examples, the light chain of theantibody includes SEQ ID NO: 28.

In some embodiments, the isolated human monoclonal antibody specificallybinds gp120, and includes a heavy chain with CDR1, CDR2, and CDR3 of anyone of SEQ ID NOs: 760-1459. In specific examples, the heavy chain ofthe human monoclonal antibody includes any one of SEQ ID NOs: 760-1459.In some embodiments, the isolated human monoclonal antibody specificallybinds gp120, and includes a heavy chain with CDR1, CDR2, and CDR3 of SEQID NO: 1316. In specific examples, the heavy chain of the humanmonoclonal antibody includes the amino acids set forth as SEQ ID NO:1316.

VRC01 and VRC03-like antibodies share unique binding site on the surfaceof gp120. Thus, in some embodiments, the antibody specifically binds toan epitope on the surface of gp120 that includes, residues 276, 278-283,365-368, 371, 455-459, 461, 469, and 472-474 of gp120 or a subset orcombination thereof (see FIGs. see FIGS. 83-91 and 93-97, the numberingof gp120 according to the HXBC2 convention is shown in FIG. 91). Withreference to FIG. 91. VRC01 and VRC0-like antibodies bind to the epitopedefined by residues N₂₇₆.T₂₇₈NNAKT₂₈₃.S₃₆₅GGD₃₆₈.I₃₇₁ . . .T₄₅₅RDGG₄₅₉.N₄₆₁.R₄₆₉.G₄₇₂GN₄₇₄ in gp120, which correspond to amino acidpositions 169, 171-176, 229-232, 235, 319-323, 325, 333, 336-338 in SEQID NO: 45. The heavy chain of a VRC01 or VRC03-like antibody can becomplemented by the light chain of VRC01, VRC02 and/or VRC03 and stillretain binding for gp120, for example retain specific binding forresidues 276, 278-283, 365-368, 371, 455-459, 461, 469, and 472-474 ofgp120 (see FIGS. 83-91 and 93-97). Thus, in some embodiments, adisclosed antibody includes the heavy chain CDRs from any one of SEQ IDNOs: 1, 3, 27, and 760-1459 and a light chain from any one of SEQ IDNOs: 2, 4, and 28 and wherein the antibody specifically binds toresidues 276, 278-283, 365-368, 371, 455-459, 461, 469, and 472-474 ofgp120. In some embodiments, a disclosed antibody includes the heavychain set forth as any one of SEQ ID NOs: 1, 3, 27, and 760-1459 and alight chain set forth as any one of SEQ ID NOs: 2, 4, and 28 and whereinthe antibody specifically binds to residues 276, 278-283, 365-368, 371,455-459, 461, 469 and 472-474 of gp120.

HIV-1 resists neutralization by most antibodies. However, the antibodyVRC01, disclosed herein, successfully neutralizes over 90% of currentcirculating HIV-1 isolates. Another antibody disclosed herein, VRC03,successfully neutralizes over 50% of current circulating HIV-1 isolates.Antibodies VRC01 and VRC03 share only about 50% sequence identity intheir variable domains, and there are a number of differences in theiractivity, including differential induction of antibody 17b binding andCCRS recognition (VRC01 induces these, whereas VRC03 does not).Nonetheless, crystal structures of VRC01 and VRC03 show virtualidentical arrangements of heavy chain and light chain recognition ofHIV-1 gp120. The similar recognition by VRC01 and VRC03 antibodies ofgp120 indicate that they are members of a class of antibodies, are ableto recognize and to neutralize HIV-1 through a similar mode of binding.Thus, these two antibodies, along with the third antibody VRC02 arerepresentative members of a class of antibodies known as VRC01-likeantibodies. Thus, disclosed herein are VRC01-like antibodies. WhileVRC01, VRC02 and VRC03 are representative members of this class ofantibodies, using 454 sequencing, 700 additional VRC01 and VRC03-likeantibodies have been identified. SEQ ID NOs: 60-759 are nucleic acidsequences encoding the heavy chains of VRC01 and VRC03 like antibodies.SEQ ID NOs: 760-1459 are amino acid sequences the heavy chains of VRC01and VRC03 like antibodies.

The heavy and light chains of VRC01 and VRC03 can be swapped for partialcomplementation .For example, the VRC01 light chain and VRC03 heavychain form active antibodies able to recognize HIV-1. In addition theVRC01 heavy chain and VRC03 light chain form active antibodies thatrecognize HIV-1. Thus, disclosed herein are VCR01-like antibodies thatcan be identified by complementation of the heavy or light chains ofVCR01 and VCR03. For example, using complementation, the heavy chainamino acid sequences of SEQ ID NOs: 760-1459 are demonstrated to beVRC01 and VRC03-like antibodies that specifically bind gp120. Analysisof these antibodies found a number of sequences, of less than 75% toeither VRC01 or VRC03 antibodies, which bioinformatics analysis indicaterecognize and neutralize HIV-1. Thus, disclosed herein is a classofantibodies, VRC01-like antibodies, that recognize and neutralizeHIV-1. Deep sequencing of heavy chains related to VRC01 in HIV-1infected individuals with high CD4-binding-site antibody titers revelessequences, for which bioinformatics analysis enables quickidentification of those which can bind and neutralize HIV-1, for exampleas a way to facilitate antibody discovery. In some embodiments, theVRC01 and VRC03-like antibodies, and other VRC01-like antibodies competewith CD4 for binding to gp120.

As disclosed herein, deep sequencing results define the variationallowed for VRC01-like recognition, and relating this to germ-line VHsequences, which delineated maturation pathways to elicit additionalneutralizing antibodies that bind to substantially similar epitopes onthe surface of gp120 in substantially the same orientation that VRC01,VRC02 and/or VRC03 bind. Thus a method is disclosed herein to determineif antibodies are in the same class specifically ising complementationof antibody H/L for function. For example, if chimeras of one antibodyheavy chain with another's light chain (and vice-versa) allows forpreservation of binding and neutralization, those two antibodiesbind/function in the same mode and thus are members of the same class.Thus, any antibody that preserves antigen binding or HIV neutralizationfunction by chain complementation with VRC01, VRC02 and/or VRC03 isconsidered a VRC01-like antibody.

The present disclosure also relates to the crystals obtained from theVRC03 or VRC01 antibody or portions thereof in complex with gp120, thecrystal structures of the VRC03 or VRC01 antibody or portions thereof incomplex with gp120, the three-dimensional coordinates of the VRC03 orVRC01 antibody or portions thereof in complex with gp120 andthree-dimensional structures of models of the VRC03 or VRC01 antibody orportions thereof in complex with gp120. The three dimensionalcoordinates of VRC01 in complex with gp120 are available at the ProteinData Bank, at accession number 3NGB, and are incorporated herein byreference in their entirety as available Jul. 7, 2010. The threedimensional coordinates of VRC03 in complex with gp120 are given inTable 1 of Provisional Application No. 61/402,314, filed Aug. 27, 2010,and are incorporated herein by reference in their entirety.

The crystal structure of the VRC03 or the VRC01 antibody in complex withgp120 provides insight for a novel binding mode for antibodies andgp120. Such a novel binding mode establishes a new class of antibodyrecognition for gp120 that is indicative of a novel class of gp120antibodies, as exemplified by VRC01, VRC02 and VRC03. Such antibodiesare termed VRC01-like antibodies. In certain embodiments a VRC01-likeantibody has a relative angle and orientation of binding of gp120 asshown in the crystal structure of the complex of the VRCR03 antibody andgp120 (see FIG. 2 d of Zhou et al., “Structural Basis for Broad andPotent Neutralization of HIV-1 by Antibody VRC01, Science 329, 811-817(2010), which is incorporated herein by reference in its entirety). Insome examples, the VRC01-like antibodies partially mimic the binding ofthe CD4 receptor, with an about 6 Å shift and an about 43 degreerotation from the CD4-defined position (see FIG. 2 d of Zhou et al.,“Structural Basis for Broad and Potent Neutralization of HIV-1 byAntibody VRC01, Science 329, 811-817 (2010), which is incorporatedherein by reference in its entirety), such as about a 45 degree rotationfrom the CD4-defined binding, for example about a 40 degree rotation,about a 50 degree rotation, about a 35 degree rotation or about a 55degree rotation. In some examples, a VRC01-like antibody is an antibodywith heavy and light chain in an orientation of heavy chain relative togp120, that differs by less than 10 about degrees, such as less thanabout 9 about degrees, less than about 8 about degrees, less than about7 about degrees, less than about 6 about degrees, less than about 5about degrees, or less than about 4 degrees and/or less than about a 5 Åtranslation from the binding angle of VRC01 and/or VRC03 to gp120, suchas less than about a 5 Å translation, such as less than about a 4 Åtranslation, than about a 3 Å translation, less than about a 2 Åtranslation, or less than about a 2 Å translation. Such a bindingcharacteristic can readily be determined from the crystal structure ofthe VRC03 or VRC01 antibody complex.

Those of skill in the art will understand that a set of structurecoordinates for the VRC01 or VRC03 antibody or portions thereof incomplex with gp120 or a portion thereof, is a relative set of pointsthat define a shape in three dimensions. Thus, it is possible that anentirely different set of coordinates could define a similar oridentical shape. Moreover, slight variations in the individualcoordinates will have little effect on overall shape. The variations incoordinates discussed above may be generated because of mathematicalmanipulations of the structure coordinates.

This disclosure further provides systems, such as computer systems,intended to generate structures and/or perform rational drug or compounddesign for an antigenic compound capable of eliciting an immune responsein a subject. The system can contain one or more or all of: atomicco-ordinate data according to VRC03 or VRC01 antibody complex or asubset thereof, and the figures derived therefrom by homology modeling,the data defining the three-dimensional structure of a VRC03 or VRC01antibody complex or at least one sub-domain thereof, or structure factordata for gp120, the structure factor data being derivable from theatomic co-ordinate data of VRC03 or VRC01 antibody complex or a subsetthereof and the figures.

In some embodiments the CDR H2 region (the C″ strand in particular)forms hydrogen-bonds to the b-15 loop of gp120. In some embodiments Asp368 of gp120 forms a salt-bridge with Arg 71 of the heavy chain. AllVRC01-like antibodies need to mimic CD4 with similar heavy chainorientations.

In some embodiments, the human monoclonal antibody specifically bindsgp41. Thus, antibodies are provided herein wherein the heavy chain ofthe antibody comprises SEQ ID NO: 5 wherein one or more of amino acids106, 107, or 109 of SEQ ID NO: 5 are substituted with a tryptophan. Inone example, only amino acid 106 of SEQ ID NO: 5 is substituted with atryptophan. In another example, only amino acid 107 is substituted for atryptophan. In an additional example, amino acids 106 and 109 aresubstituted with a tryptophan. In some embodiments, the human monoclonalantibody comprises the heavy chain amino acid sequence set forth as SEQID NOs: 6, 7, 8 or 9.

In a further example, the human monoclonal antibody specifically bindsgp41, and includes a heavy chain comprising the amino acid sequence setforth as SEQ ID NO: 5 wherein one or more of amino acids 106, 107, or109 of SEQ ID NO: 5 are substituted with a tryptophan, and includes alight chain that includes the amino acid sequence set forth as SEQ IDNO: 10.

The heavy and the light chain are selected so that the antibodyspecifically binds either gp120 or gp41. In one example, the antibodyspecifically binds gp120 and includes the CDRs of the heavy chain aminoacid sequence set forth as one of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ IDNO: 27 or any one of SEQ ID NOs: 760-1459 and the CDRs of the lightchain amino acid sequence set forth as one of SEQ ID NO: 2, SEQ ID NO: 4or SEQ ID NO: 28. Thus, the antibody can specifically bind gp120 andincludes the heavy chain amino acid sequence set forth as one of SEQ IDNO: 1, SEQ ID NO: 3 or SEQ ID NO: 27 or any one of SEQ ID NOs: 760-1459,and the light chain amino acid sequence set forth as one of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 28.

In another example, the antibody specifically binds gp41. Thus, theantibody can include the CDRs of the heavy chain amino acid sequence setforth as one of SEQ ID NO: 6-9 and the CDRs of the light chain aminoacid sequence set forth as one of SEQ ID NO: 10. The antibody caninclude the heavy chain amino acid sequence set forth as one of SEQ IDNOs: 6-9 and the light chain amino acid sequence set forth as SEQ ID NO:10.

Fully human monoclonal antibodies include human framework regions. Thus,any of the antibodies that specifically bind gp120 or gp41 herein caninclude the human framework region and can include the framework regionsof the amino acid sequence set forth in one of SEQ ID NOs: 1-10, 27 and28. However, the framework regions can be from another source.Additional examples of framework sequences that can be used include theamino acid framework sequences of the heavy and light chains disclosedin PCT Publication No. WO 2006/074071 (see, for example, SEQ ID NOs:1-16), which is herein incorporated by reference.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁or an IgG₂.The class of an antibody that specifically binds gp120 or gp41 can beswitched with another. In one aspect, a nucleic acid molecule encodingV_(L) or V_(H) is isolated using methods well-known in the art, suchthat it does not include any nucleic acid sequences encoding theconstant region of the light or heavy chain, respectively.

In particular examples, the V_(H) amino acid sequence is SEQ ID NOs: 1,3, 5, 6, 7, 8, 9, 27 or any one of 760-1459. In other examples, theV_(L) amino acid sequence is SEQ ID NOs: 2, 4, 6, 10 or 28. In furtherexamples, the V_(H) nucleic acid sequence is SEQ ID NOs: 29, 31 or 33.In other examples, the V_(L) nucleic acid sequence is SEQ ID NOs: 30, 32or 34. The nucleic acid molecule encoding V_(L) or V_(H) is thenoperatively linked to a nucleic acid sequence encoding a C_(L) or C_(H)from a different class of immunoglobulin molecule. This can be achievedusing a vector or nucleic acid molecule that comprises a C_(L) or C_(H)chain, as known in the art. For example, an antibody that specificallybinds gp120, such as VRC01, VRC02 or VRC03, that was originally IgM maybe class switched to an IgG. Similar class switches can also be achievedwith an antibody that specifically binds gp41, such as 2F5, or any ofits variants described herein. Class switching can be used to convertone IgG subclass to another, such as from IgG₁ to IgG₂.

In some examples, the disclosed and antibodies are oligomers ofantibodies, such as dimers trimers, tetramers, pentamers, hexamers,septamers, octomers and so on. In some examples, the antibodies arepentamers, for example pentamers of VRC01, VRC03, VRC02, or VRC01 andVRC03 like antibodies. Is a specific example the antibody is a apentameric IgM antibody carrying the VRC01 V region (see e.g. FIG. 92).

By definition, the CDRs of the light chain are bounded by the residuesat positions 24 and 34 (L-CDR1), 50 and 56 (L-CDR2), 89 and 97 (L-CDR3);the CDRs of the heavy chain are bounded by the residues at positions 31and 35b (H-CDR1), 50 and 65 (H-CDR2), 95 and 102 (H-CDR3), using thenumbering convention delineated by Kabat et al., (1991) Sequences ofProteins of Immunological Interest, 5^(th) Edition, U.S. Department ofHealth and Human Services, Public Health Service, National Institutes ofHealth, Bethesda, Md. (NIH Publication No. 91-3242, which isspecifically incorporated herein by reference in its entirety).

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and specifically bind gp120 or gp41. These antibodyfragments retain the ability to selectively bind with the antigen. Thesefragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of m912.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. In particularexamples, the V_(H) amino acid sequence includes the CDRs from one ofSEQ ID NOs: 1, 3, 5, 6, 7, 8, 9 or 27, or any one of 760-1459. In otherexamples, the V_(L) amino acid sequence includes the CDRs from SEQ IDNOs: 2, 4, 6 or 28. In additional examples, the V_(H) amino acidsequence includes the amino acid sequence set forth as one of SEQ IDNOs: 1, 3, 5, 6, 7, 8, 9 or 27, or any one of 760-1459. In otherexamples, the V_(L) amino acid sequence includes the amino acid sequenceset forth as SEQ ID NOs: 2, 4, 6 or 28. In further examples, the V_(H)nucleic acid sequence is SEQ ID NOs: 29, 31 or 33. In other examples,the V_(L) nucleic acid sequence is SEQ ID NOs: 30, 32 or 34.

If the V_(H) and the V_(L) are expressed non-contiguously, the chains ofthe Fv antibody are typically held together by noncovalent interactions.However, these chains tend to dissociate upon dilution, so methods havebeen developed to crosslink the chains through glutaraldehyde,intermolecular disulfides, or a peptide linker. Thus, in one example,the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chainvariable region and the light chain variable region are chemicallylinked by disulfide bonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. In particular examples, the V_(H) sequence is SEQ IDNOs: 1, 3, 5, 6, 7, 8, 9, 27 or any one of SEQ ID NOs: 760-1459. Inother examples, the V_(L) sequence is SEQ ID NOs: 2, 4, 6 or 28.Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The antibodies disclosed herein can be isolated using cloaked antigens,as described in PCT Publication No. WO 2009/100376. Briefly, antigensare cloaked to target antigenicity of the antigen to a specific epitopethat specifically bound by the antibody of interest, such as aneutralizing antibody.

In some embodiments, the isolated antigens used for antibody isolationinclude a target epitope defined by atomic coordinates of those aminoacids of the antigen that contact an antibody of interest thatspecifically binds the antigen. The gp120 and pg41 antigens have beenmodified to substitute the surface exposed amino acids located exteriorto the target epitope of gp41 or gp120 to focus the antigenicity of theantigen to the target epitope. For example, the method can removenon-target epitopes that might interfere with specific binding of anantibody to the target epitope on gp120 or gp41. In some examples, theamino acid substitutions result in the antigen not being bound byantibodies in a polyclonal serum that specifically bind surface exposedamino acid residues of the wild-type antigen located exterior of thetarget epitope. In some embodiments, the amino acid substitutions alterantigenicity of the antigen in vivo as compared to the wild-type antigen(unsubstituted antigen) but do not introduce additional glycosylationsites as compared to the wild-type antigen. In some embodiments, thatantigen is glycosylated. In some embodiments the cloaked antigen ismodified to substitute one or more residues recognized by the antibodyof interest to abolish antigen recognition. In some examples, abiotinylation peptide (for example SEQ ID NO: 26) can be fused to thecloaked antigen. Biotinylated cloaked antigen can then be used to stainand thus identify cells, such as PBMC, expressing an antibody ofinterest.

Additional recombinant human neutralizing antibodies that specificallybind the same epitope of gp120 bound by the antibodies disclosed hereinthat specifically bind gp120 (for example, the epitope of gp120specifically bound by Vc2a11 (VRC01) and Vc2a34 (VRC02), or the specificepitope bound by VRC03, see the examples section below), or humanneutralizing antibodies that specifically the same epitope of gp41 boundby antibodies that specifically bind gp41 (for example, the epitopebound by L_(100A)W, F_(100B)W, V_(100D)W, and L_(100A)W-V_(100D)Wvariants of antibody 2F5, see the Examples section below), can beisolated by screening of a recombinant combinatorial antibody library,such as a Fab phage display library (see, for example, U.S. PatentApplication Publication No. 2005/0123900). In some cases the phagedisplay libraries are prepared using cDNAs of the variable regions ofheavy and light chains prepared from mRNA derived from humanlymphocytes. Methodologies for preparing and screening such librariesare known in the art. There are commercially available kits forgenerating phage display libraries (for example, the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There arealso other methods and reagents that can be used in generating andscreening antibody display libraries (see, for example, U.S. Pat. No.5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; Fuchs et al., Bio/Technology9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992;Huse et al., Science 246:1275-1281, 1989; McCafferty et al., Nature348:552-554, 1990; Griffiths et al., EMBO J. 12:725-734, 1993)

In one embodiment, to isolate additional human antibodies thatspecifically bind either gp120 or gp41, a neutralizing antibody thatspecifically binds gp120 or gp41, as described herein, is first used toselect human heavy and light chain sequences having similar bindingactivity toward gp120 or gp41, such as using the epitope imprintingmethods disclosed in PCT Publication No. WO 93/06213. The antibodylibraries used in this method are scFv libraries prepared and screened,using methods such as those as described in PCT Publication No. WO92/01047, McCafferty et al., Nature 348:552-554, 1990; and/or Griffithset al., EMBO J. 12:725-734, 1993 using gp120.

Once initial human variable light chain (V_(L)) and variable heavy chain(V_(H)) segments are selected, “mix and match” experiments, in whichdifferent pairs of the initially selected V_(L) and V_(H) segments arescreened for gp120 or gp41 binding, such as to the epitopes bound byVRC01, VRC02, and VRC03 (gp120) or to the epitope bound by theL_(100A)W, F_(100B)W, V_(100D)W, and L_(100A)W-V_(100D)W variants ofantibody 2F5 (in gp41) are performed to select V_(L)/V_(H) paircombinations of interest. Additionally, to increase binding affinity ofthe antibody, the V_(L) and V_(H) segments can be randomly mutated, suchas within H-CDR3 region or the L-CDR3 region, in a process analogous tothe in vivo somatic mutation process responsible for affinity maturationof antibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying V_(H) and V_(L) regionsusing PCR primers complementary to the H-CDR3 or L-CDR3, respectively.In this process, the primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be tested to determine thebinding affinity for gp120 or gp41. In particular examples, the V_(H)amino acid sequence is SEQ ID NOs: 1, 3, 5, 6, 7, 8, 9, 27, or any oneof 760-1459. In other examples, the V_(L) amino acid sequence is SEQ IDNOs: 2, 4, 6 or 28.

Following screening and isolation of an antibody that binds gp120 orgp41 from a recombinant immunoglobulin display library, nucleic acidencoding the selected antibody can be recovered from the display package(for example, from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques, as described herein. Ifdesired, the nucleic acid can be further manipulated to create otherantibody fragments, also as described herein. To express a recombinantantibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described herein.

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibody or portion thereof is derivatized such that thebinding to gp120 or gp41 is not affected adversely by the derivatizationor labeling. For example, the antibody can be functionally linked (bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (forexample, a bispecific antibody or a diabody), a detection agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company (Rockford, Ill.).

An antibody that specifically binds gp120 or gp41 can be labeled with adetectable moiety. Useful detection agents include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein, Yellow fluorescent protein. Anantibody can also be labeled with enzymes that are useful for detection,such as horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it can be detected by adding additionalreagents that the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase is presentthe addition of hydrogen peroxide and diaminobenzidine leads to acolored reaction product, which is visually detectable. An antibody mayalso be labeled with biotin, and detected through indirect measurementof avidin or streptavidin binding. It should be noted that the avidinitself can be labeled with an enzyme or a fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes.Examples of labels for polypeptides include, but are not limited to, thefollowing radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

B. Polynucleotides and Expression

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies) can readily be produced by one of skill in the art. Forexample, these nucleic acids can be produced using the amino acidsequences provided herein (such as the CDR sequences, heavy chain andlight chain sequences), sequences available in the art (such asframework sequences), and the genetic code. The nucleic acids encodingthe VRC01 heavy chain was deposited as ATCC Deposit Number PTA-10412;the nucleic acids encoding the VRC01 light chain was deposited as ATCCDeposit Number PTA-10411; the nucleic acids encoding the VRC02 heavychain was deposited as ATCC Deposit Number PTA-10414; and the nucleicacidd encoding the VRC02 light chain was deposited as ATCC DepositNumber PTA-10413. Nucleic acids encoding the VRC03 heavy chain and VRC03light chain were deposited at the ATCC on Dec. 23, 2009 as ATCC DepositNumbers PTA-10551 and PTA-10550, respectively. All deposits were made inaccordance with the Budapest Treaty.

V_(H) nucleic acid sequences are set forth as SEQ ID NOs: 29, 31, 33,and any one of 61-759 and include degenerate variants thereof. V_(L)nucleic acid sequences are set forth as SEQ ID NOs: 30, 32 and 34, andinclude degenerate variants thereof. One of skill in the art can readilyuse the genetic code to construct a variety offunctionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same antibody sequence, or encode a conjugate or fusionprotein including the V_(L) and/or V_(H) nucleic acid sequence.

Nucleic acid sequences encoding the antibodies that specifically bindgp120 or gp41 can be prepared by any suitable method including, forexample, cloning of appropriate sequences or by direct chemicalsynthesis by methods such as the phosphotriester method of Narang etal., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brownet al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramiditemethod of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solidphase phosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., supra, Berger and Kimmel (eds.), supra, andAusubel, supra. Product information from manufacturers of biologicalreagents and experimental equipment also provide useful information.Such manufacturers include the SIGMA Chemical Company (Saint Louis,Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway,N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem GenesCorp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc.,GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

Any of the nucleic acids encoding any of the antibodies, V_(H) and/orV_(L), disclosed herein (or fragment thereof) can be expressed in arecombinantly engineered cell such as bacteria, plant, yeast, insect andmammalian cells. These antibodies can be expressed as individual V_(H)and/or V_(L) chain, or can be expressed as as a fusion protein. Animmunoadhesin can also be expressed. Thus, in some examples, nucleicacids encoding a V_(H) and V_(L), and immunoadhesin are provided. Thenucleic acid sequences can optionally encode a leader sequence.

To create a single chain antibody, (scFv) the V_(H)- and V_(L)-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃,such that the V_(H) and V_(L) sequences can be expressed as a contiguoussingle-chain protein, with the V_(L) and V_(H) domains joined by theflexible linker (see, e.g., Bird et al., Science 242:423-426, 1988;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCaffertyet al., Nature 348:552-554, 1990). Optionally, a cleavage site can beincluded in a linker, such as a furin cleavage site.

The nucleic acid encoding the V_(H) and/or the V_(L) optionally canencode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM orIgG Fc domain. The Fc domain can be an optimized Fc domain, as describedin U.S. Published Patent Application No. 20100/093979, incorporatedherein by reference. In one example, the immunoadhesin is an IgG₁ Fc.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to gp120and to another molecule, such as gp41. The encoded V_(H) and V_(L)optionally can include a furin cleavage site between the V_(H) and V_(L)domains.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of proteinsincluding E. coli, other bacterial hosts, yeast, and various highereukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

The host cell can be a gram positive bacteria including, butare notlimited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Methods for expressing protein in gram positivebacteria, such as Lactobaccillus are well known in the art, see forexample, U.S. Published Patent Application No. 20100/080774. Expressionvectors for lactobacillus are described, for example in U.S. Pat. No.6,100,388, and U.S. Pat. No. 5,728,571. Leader sequences can be includedfor expression in Lactobacillus. Gram negative bacteria include, but notlimited to, E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, andUreaplasma.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA or cDNA to a promoter(which is either constitutive or inducible), followed by incorporationinto an expression cassette. The promoter can be any promoter ofinterest, including a cytomegalovirus promoter and a human T celllymphotrophic virus promoter (HTLV)-1. Optionally, an enhancer, such asa cytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosmal binding sequences), and atranscription/translation terminator. For E. coli, this includes apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the immunoconjugates, effectormoieties, and antibodies of the present disclosure can also beconstructed in whole or in part using standard peptide synthesis wellknown in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of antibodies and/or refolding to an appropriateactive form, including single chain antibodies, from bacteria such as E.coli have been described and are well-known and are applicable to theantibodies disclosed herein. See, Buchner et al., Anal. Biochem.205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al.,Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

C. Isolation of Additional Antibodies with Enhanced Binding Properties

Epitope scaffolds have been used to isolate antibodies with particularbinding specificity (See PCT Publication No. WO 2008/025015). Briefly,an epitope, such as an epitope of a pathogenic agent (for example, anepitope of an HIV-1 polypeptide) recognized by broadly neutralizingantibodies is placed into an appropriate peptide scaffold that preservesits structure and antigenicity. Such epitope scaffolds can then be usedas an immunogen to elicit an epitope-specific antibody response in asubject. In another example, such scaffolds can be used to identifyspecific serum reactivities against the target epitope of the scaffold.This scaffolding technology is applicable not only to HIV-1, but to anypathogen for which a broadly neutralizing antibody and its respectiveepitope has been characterized at the atomic-level.

The design of epitope-protein scaffolds which elicit selectedneutralizing antibodies is disclosed in PCT Publication No. WO2008/025015, which is incorporated herein by reference. In general, theprotocols utilize searchable databases containing the three dimensionalstructure of proteins, epitopes, and epitope-antibody complexes toidentify proteins that are capable of structurally accommodating atleast one selected epitope on their surface. Protein folding energeticpredictions are further utilized to make energetic predictions. Thepredicted energies may be used to optimize the structure of theepitope-scaffold and filter results on the basis of energy criteria inorder to reduce the number of candidate proteins and identifyenergetically stable epitope-scaffolds.

In one embodiment, a “superposition” epitope-scaffold can be designedand utilized. Superposition epitope-scaffolds are based upon scaffoldproteins having an exposed segment on their surface with a similarconformation as a selected target epitope. The backbone atoms in thissuperposition region can be structurally superimposed onto the targetepitope with less than a selected level of deviation from their nativeconfiguration. Candidate scaffolds are identified by computationallysearching through a library of three-dimensional structures. Thecandidate scaffolds are further designed by putting epitope residues inthe superposition region of the scaffold protein and making additionalmutations on the surrounding surface of the scaffold to preventundesirable interactions between the scaffold and the epitope or thescaffold and the antibody.

Superposition is advantageous in that it is a conservative technique.Epitope-scaffolds designed by superposition require only a limitednumber of mutations on the surface of known, stable proteins. Thus, thedesigns can be produced rapidly and a high fraction of the first rounddesigns are likely to fold properly.

In another embodiment, “grafting” epitope scaffolds are utilized.Grafting epitope scaffolds utilize scaffold proteins that canaccommodate replacement of an exposed segment with the crystallizedconformation of the target epitope. For each suitable scaffoldidentified by computationally searching through a database of knownthree-dimensional structures, an exposed segment is replaced by thetarget epitope. The surrounding protein side chains are further mutatedto accommodate and stabilize the inserted epitope. Mutations are furthermade on the surface of the scaffold to avoid undesirable interactionsbetween the scaffold and epitope or scaffold and antibody. Graftingepitope-scaffolds should substantially mimic the epitope-antibodyinteraction, as the epitope is presented in substantially its nativeconformation. As such, grafting may be utilized to treat complexepitopes which are more difficult to incorporate using superpositiontechniques.

In certain embodiments, protein and design calculations are performedusing the ROSETTA™ computer program to design the eptiope scaffolds.ROSETTA™ is a software application, developed at least in part at theUniversity of Washington which provides protein structure predictions.ROSETTA™ utilizes physical models of the macromolecular interactions andalgorithms for finding the lowest energy structure for an amino acidsequence in order to predict the structure of a protein. FurthermoreROSETTA™ may use these models and algorithms to find the lowest energyamino acid sequence for a protein or protein-protein complex for proteindesign. The ROSETTA™ energy function and several modules of the ROSETTA™protein structure modeling and design platform are employed in theprotein scaffold design discussed below.

Described herein are methods of increasing an antibody binding affinityand neutralizing capacity that utilize this epitope scaffoldingtechnology. In the methods described herein, an original (parental)antibody that specifically binds a scaffolded epitope is identified andsequenced. The amtibody bining determinants of antibody reactivity arethen identified by mutagenesis (for example, amino acid substitutions)of the antibody sequences, wherein variant antibodies are produced.These amino acid substitutions can be made in one or more CDRs and/or inone or more framework regions of the original antibody. The amino acidsubstitutions can be a replacement of the amino acid in the originalantibody for a tryptophan. In some embodiments, the antibodies includeat most one, at most two, at most three or at most four amino acidssubstitutions, such as in the CDRs. These variant antibodies, such asthe antibodies including one, two, three or four amino acidssubstitutions, are then evaluated for binding to the epitope scaffold.Antibodies are selected that have altered binding affinity for theepitope scaffold as compared to the original (parental) antibody.

In particular examples, selection of residues for mutagenesis is aidedby structural modeling of the scaffold-antibody interaction. To producean antibody with enhanced binding affinity, the amino acid(s) that havebeen identified as critical for antibody reactivity are then furthersubstituted and the effects on antibody reactivity measured by furtherprobing with the epitope scaffold.

Any method known to the art can be used to determine antibody-scaffoldaffinity. In some examples, the epitope scaffold probe is fused to abiotinylation peptide. In particular examples, the biotinylation peptideis SEQ ID NO: 26.

In some examples, the amino acid residues in the antibody that areresponsible for specific binding to the epitope are indicated by adecrease in antibody affinity of the variant antibody as compared to theparental antibody. In some embodiments, antibodies are selected whereinbinding is decreased by at least 20%, at least 30% at least 40% at least50% at least 100% (2-fold), at least 200%, at least 300%, at least 400%,at least 500%, at least 600%, at least 700%, at least 800%, at least900%, or at least 1,000% (10-fold) as compared to the original antibody.The decrease of affinity for the scaffold identifies the variantantibody as compared to the parental antibody to identify the one ormore amino acids as critical for antigen binding. In one example, thecomplete loss of antibody binding affinity for the epitope scaffoldidentifies the one or more amino acid residues as critical for specificbinding of antibody to the epitope.

In other embodiments, variant antibodies are selected wherein binding isincreased by at least 20%, at least 30% at least 40% at least 50% atleast 100% (2-fold), at least 200%, at least 300%, at least 400%, atleast 500%, at least 600%, at least 700%, at least 800%, at least 900%,or at least 1,000% (10-fold) as compared to the parental antibody. Anincrease of the binding of the variant antibody as compared to theparental antibody identifies the one or more amino acid residues ascritical for specific binding of the antibody to the epitope.

An exemplary method is disclosed herein (see the EXAMPLES section),wherein this technology is utilized for an antibody that specificallybinds an antigenic glycoprotein of HIV. However, this method is broadlyapplicable to antibodies that specifically bind any antigen of interest.In some embodiments, the antibody specifically binds a pathogen ofinterest. Pathogens include viruses, fungi, bacteria, and protozoa. Inother example the antibody specifically binds a tumor antigen ofanteerest.

While this disclosure is written with specific reference to theidentification of antibodies that are specific for HIV, such asantibodies specific for gp120 and gp140 from HIV, the methods disclosedherein, including those described in the EXAMPLES are equally applicableto the identification of other antigens, for example antigens frompathogenic sources as well as tumor antigens. In some examples, usingthe epitope scaffolds such as described in PCT Publication No. WO2008/025015 can be used to identify epitope specific B-cells and isolatespecific IgG clones that bind to a target epitope, such as the site ofvulnerability ion the surface of gp120, as disclosed herein. Withreference to FIG. 117, in some examples, a subject is selected thatproduces, or has broadly neutralizing sera, such that the B-cellsisolated from that subject are believed express one or more broadlyneutralizing antibodies to an antigen of interest, such as an antigenfrom a pathogenic organism or a tumor (see FIG. 117, 1). B-cells areisolated from the subject, for example using the procedures outlined inthe EXAMPLES section, and the isolated B-cells are contacted with atarget antigen of interest (see FIG. 117, 2), such as a resurfacedantigen, and the complex of the B-cells and the target antigen ofinterest is isolated (see FIG. 117, 3). Nucleic acids are obtained fromthe B-cells are analyzed and antibodies encoded by the Ig gene aresynthesized (see FIG. 117, 4) and the antibodies are furthercharacterized (see FIG. 117, 5). In some examples, the antibody antigencomplexes are further characterized structurally, for example usingX-ray diffraction methods (see FIG. 117, 6), which allows the importantantibody/antigen contacts to be mapped. This information can be used todefine classes of neutralizing antibodies specific for an antigen ofinterest, for example as is disclosed herein for the class of VRC01 andVRC03-like antibodies. The structural information about theantigen/antibody contacts and conformation can be analyzed inconjunction with sequencing data, such as 454 sequencing data, toidentify additional antibodies that have the same or similar bindingproperties, in that they are highly specific for a specific neutralizingepitope on the surface of the antigen of interest. By combining sequenceanalysis, such as 454 sequencing with structural characterization ofantibody/antigen interactions at the atomic level it is now possible toidentify classes of neutralizing antibodies from a subject. As disclosedherein, this has now been demonstrated for HIV using designed gp120antigens. In other words, the combination of sequencing, such as 454sequencing with identified binding motifs in antibodies allows theidentification of additional antibodies. Importantly, however, it allowsfor a short-cut (see e.g. steps 1-7 in FIG. 117) as these antibodies aredirectly identified from B-cells as these antibodies are directlyidentified from B-cells without the requirement for isolating antigenspecific B cells. In doing so, it ties genomics technologies directly tosera characterization. This tie permits direct interrogation of theantibodyome, which is the family of antibodies specific for an antigenor even an organism or cancer, or interest. In some examples, themethods described herein can be used to examine a time course ofantibody maturation from seroconversion to production of broadlyneutralizing antibodies. In some embodiments, the methods describedherein are used to monitor the development of antibodies in vaccines isa subject, for example to allow feedback at the antibody sequence leveland subsequent redesign of the vaccines during vaccine development.

The methods disclosed herein have broad applications for of identifyingspecific antibodies, classes or species of antibodies with definedspecificity, for example as exemplified by the identification of VRC01and VRC03-like antibodies disclosed herein. This combination ofstructural and genomic analysis of 1g may provide a generic way ofidentifying specific antibodies, as well as classes or species ofantibodies with defined specificities. Such antibodies, like VRC01 andrelated antibodies, can potentially be used for prevention strategies,such as microbicides or passive protection of HIV infection, vaccinedesign, diagnostics, and therapy of infected individuals.

Thus, viral antigenic epitopes can be used with the methods disclosedherein to identify classes of antibodies specific for the antigen ofinterest. Antigens of use in the methods disclosed herein include, butare not limited to, antigenic epitopes from dengue virus, humanimmunodeficiency virus, influenza virus, metapneumovirus, norovirus,papillomavirus, parvovirus, SARS virus, smallpox virus, picornaviruses,respiratory syncitial virus, parainfluenza virus, measles, hepatitis,measles, varicella zoster, rabies and West Nile virus, among manyothers. In some embodiments, the antigenic epitope is from a viruscauses a respiratory disorder (for example, adeno, echo, rhino,coxsackie, influenza, parainfluenza, or respiratory syncytial virus), adigestive disorder (for example, rota, parvo, dane particle, orhepatitis A virus), an epidermal-epithelial disorder (for example,verruca, papilloma, molluscum, rubeola, rubella, small pox, cowpox), aherpes virus disease (for example, varicella-zoster, simplex I, orsimplex II virus), an arbovirus disease (for example, dengue, yellow, orhemorrhagic fevers), a viral disease of the central nervous system (forexample, polio or rabies), a viral heart disease, or acquired immunedeficiency (AIDS). The antigenic epitope can also be from a bacteria. Insome examples, bacteria antigenic epitope is a Pyogenic cocci antigenfrom an organism that causes, for example, staphylococcal,streptococcal, pneumococcal, meningococcal, and gonococcal infections; agram-negative rod antigen from an organism that causes, for example, E.coli, Klebsiella, enterobacter, pseudomonas, or legionella infections;an antigenic epitope for an organism that causes, for example,hemophilus influenza, bordetella pertussis, or diphtheria infections.Also encompassed in this disclosure are bacterial antigens fromenteropathic bacteria (for example, S. typhi), clostridia (for example,C. tetani or C. botulinum)), and mycobacteria (for example, M.tuberculosis or M. leprae). Exemplary antigens are the CFP10 polypeptideor a domain of other polypeptides of Mycobacterium tuberculosis, or of adomain of the pilus polypeptide of Vibrio cholera, the CjaA polypeptideof Campylobacter coli, the Sfb 1 polypeptide of Streptococcus pyogenes,the UreB polypeptide Helicobacter pylori, or of other pathogenicorganisms such as the circumsporozoite polypeptide of Plasmodiumfalciparum. Non-limiting examples of bacterial (including mycobacterial)epitopes can be found, for example, in Mei et al., Mol. Microbiol.26:399-407, 1997; and U.S. Pat. No. 6,790,950 (gram negative bacteria);U.S. Pat. No. 6,790,448 (gram positive bacteria); U.S. Pat. Nos.6,776,993 and 6,384,018 (Mycobacterium tuberculosis).

In additional examples, the antigenic epitope is from a Chlamydia thatcauses ornithosis (C. psittaci), chlamydial urethritis and cervicitis(C. trachomatis), inclusion conjunctivitis (C. trachomatis), trachoma(C. trachomatis), or lymphogranuloma venereum (C. trachomatis)). Inadditional examples, the antigen epitope is from rickettsia that causestyphus fever (R. prowazekii), Rocky Mountain spotted fever (R.rickettsi), scrub fever (R. tsutsugamushi), or Q fever (Coxiellaburnetii).

In particular embodiments, the antigenic epitope is from a fungus, suchas Candidae (for example, C. albicans) or Aspergillis (for example, A.fumigatus). In other embodiments, the protozoan antigen is from, forexample, Giardia Lamblia, Trichomoniasis, Pneumocystosis, Plasmodium,Leishmania, or Toxoplasma. In further embodiments, the helminth antigenis from, for example, Trichuris, Necator americanus (hookworm disease),Ancylostoma duodenale (hookworm disease), Trichinella spiralis, or S.mansoni.

In additional embodiments, the method is applied to identify antibodiesthat bind antigenic epitopes of tumor antigens. Tumor antigens include,but are not limited to carcinoembryonic antigen (“CEA:” e.g., GENBANK®Accession No. AAA62835), ras proteins (see, e.g., Parada et al. Nature297:474-478, 1982), p53 protein (e.g., GENBANK® Accession No. P07193),prostate-specific antigen (“PSA:” e.g., GENBANK® Accession Nos.NP001639, NP665863), Mucl (e.g., GENBANK® Accession No. P15941),tyrosinase (see, e.g., Kwon et al., Proc Natl Acad Sci USA 84:7473-7477,1987, erratum Proc Natl Acad Sci USA 85:6352, 1988 Melanoma-associatedantigen (MAGES: for examples, see, U.S. Pat. Nos. 5,462,871; 5,554,724;5,554,506; 5,541,104 and 5,559,558,995). The tumor antigen can be from atumor of any organ or tissue, including but not limited to solid organtumors. For example, the tumor can be melanoma, colon-, breast-, lung,cervical-, ovarian, endometrial-, prostate-, skin-, brain-, liver-,kidney, thyroid, pancreatic, esophageal-, or gastric cancer, leukemias,lymphomas, multiple myeloma, myelodysplastic syndrome, premalignanthuman papiloma virus (HPV)-related lesions, intestinal polyps and otherchronic states associated with increased tumor risk.

D. Compositions and Therapeutic Methods

Methods are disclosed herein for the prevention or treatment of an HIVinfection, such as an HIV-1 infection. Preention can include inhibitionof infection with HIV-1. The methods include contacting a cell with aneffective amount of the human monoclonal antibodies disclosed hereinthat specifically binds gp120 or gp41, or a functional fragment thereof.The method can also include administering to a subject a therapeuticallyeffective amount of the human monoclonal antibodies to a subject.

Methods to assay for neutralization activity include, but are notlimited to, a single-cycle infection assay as described in Martin et al.(2003) Nature Biotechnology 21:71-76. In this assay, the level of viralactivity is measured via a selectable marker whose activity isreflective of the amount of viable virus in the sample, and the IC50 isdetermined. In other assays, acute infection can be monitored in the PM1cell line or in primary cells (normal PBMC). In this assay, the level ofviral activity can be monitored by determining the p24 concentrationsusing ELISA. See, for example, Martin et al. (2003) Nature Biotechnology21:71-76.

HIV infection does not need to be completely eliminated for thecomposition to be effective. For example, a composition can decrease HIVinfection by a desired amount, for example by at least 10%, at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100% (elimination ofdetectable HIV infected cells), as compared to HIV infection in theabsence of the composition. In example, the cell is also contacted withan effective amount of an additional agent, such as anti-viral agent.The cell can be in vivo or in vitro. The methods can includeadministration of one on more additional agents known in the art. Inadditional examples, HIV replication can be reduced or inhibited bysimilar methods. HIV replication does not need to be completelyeliminated for the composition to be effective. For example, acomposition can decrease HIV replication by a desired amount, forexample by at least 10%, at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100% (elimination of detectable HIV), as compared to HIVreplication in the absence of the composition. In one example, the cellis also contacted with an effective amount of an additional agent, suchas anti-viral agent. The cell can be in vivo or in vitro.

Compositions are provided that include one or more of the antibodiesthat specifically bind gp120 or gp41, or functional fragments thereof,that are disclosed herein in a carrier. The compositions can be preparedin unit dosage forms for administration to a subject. The amount andtiming of administration are at the discretion of the treating physicianto achieve the desired purposes. The antibody can be formulated forsystemic or local administration. In one example, the antibody thatspecifically binds gp120 or gp41 is formulated for parenteraladministration, such as intravenous administration.

The compositions for administration can include a solution of theantibody that specifically binds gp120 or the antibody that specificallybinds gp41 dissolved in a pharmaceutically acceptable carrier, such asan aqueous carrier. A variety of aqueous carriers can be used, forexample, buffered saline and the like. These solutions are sterile andgenerally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofantibody in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

A therapeutically effective amount of a human gp120-specific antibody orhuman gp41-specific antibody will depend upon the severity of thedisease and/or infection and the general state of the patient's health.A therapeutically effective amount of the antibody is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver. These compositions can be administered in conjunction withanother therapeutic agent, either simultaneously or sequentially.

In one embodiment, administration of the antibody results in a reductionin the establishment of HIV infection and/or reducing subsequent HIVdisease progression in a subject. A reduction in the establishment ofHIV infection and/or a reduction in subsequent HIV disease progressionencompass any statistically significant reduction in HIV activity. Insome embodiments, methods are disclosed for treating a subject with anHIV-1 infection. These methods include administering to the subject atherapeutically effective amount of an antibody, or a nucleic acidencoding the antibody, thereby preventing or treating the HIV-1infection.

Studies have shown that the rate of HIV transmission from mother toinfant is reduced significantly when zidovudine is administered toHIV-infected women during pregnancy and delivery and to the offspringafter birth (Connor et al., 1994 Pediatr Infect Dis J 14: 536-541).Several studies of mother-to-infant transmission of HIV havedemonstrated a correlation between the maternal virus load at deliveryand risk of HIV transmission to the child. The present disclosureprovides isolated human monoclonal antibodies that are of use indecreasing HIV-transmission from mother to infant. Thus, in someexamples a therapeutically effective amount of a human gp120-specificantibody or human gp41-specific antibody is administered in order toprevent transmission of HIV, or decrease the risk of transmission ofHIV, from a mother to an infant. In some examples, a therapeuticallyeffective amount of the antibody is administered to mother and/or to thechild at childbirth. In other examples, a therapeutically effectiveamount of the antibody is administered to the mother and/or infant priorto breast feeding in order to prevent viral transmission to the infantor decrease the risk of viral transmission to the infant. In someembodiments, both a therapeutically effective amount of the antibody anda therapeutically effective amount of another agent, such as zidovudine,is administered to the mother and/or infant.

For any application, the antibody can be combined with anti-retroviraltherapy. Antiretroviral drugs are broadly classified by the phase of theretrovirus life-cycle that the drug inhibits. The disclosed antibodiescan be administered in conjunction with Nucleoside and nucleotidereverse transcriptase inhibitors (nRTI), Non-nucleoside reversetranscriptase inhibitors (NNRTI), Protease inhibitors, Entry inhibitors(or fusion inhibitors), Maturation inhibitors, or a Broad spectruminhibitors, such as natural antivirals. Exemplary agents includelopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabineand efavirenz.

Single or multiple administrations of the compositions including theantibodies disclosed herein are administered depending on the dosage andfrequency as required and tolerated by the patient. In any event, thecomposition should provide a sufficient quantity of at least one of theantibodies disclosed herein to effectively treat the patient. The dosagecan be administered once but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

E. Diagnostic Methods and Kits

A method is provided herein for the detection of the expression of gp120or gp41 in vitro or in vivo. In one example, expression of gp120 or gp41is detected in a biological sample, and can be used to detect HIV-1infection. The sample can be any sample, including, but not limited to,tissue from biopsies, autopsies and pathology specimens. Biologicalsamples also include sections of tissues, for example, frozen sectionstaken for histological purposes. Biological samples further include bodyfluids, such as blood, serum, plasma, sputum, spinal fluid or urine.

In several embodiments, a method is provided for detecting AIDS and/oran HIV-1 infection in a subject. The disclosure provides a method fordetecting HIV-1 in a biological sample, wherein the method includescontacting a biological sample with the antibody under conditionsconducive to the formation of an immune complex, and detecting theimmune complex, to detect the gp120 or gp41 in the biological sample. Inone example, the detection of gp120 or gp41 in the sample indicates thatthe subject has an HIV infection. In another example, the detection ofgp120 or gp41 in the sample indicates that the subject has AIDS. Inanother example, detection of gp120 or gp41 in the sample confirms adiagnosis of AIDS and/or an HIV-1 infection in a subject.

In some embodiments, the disclosed antibodies are used to test vaccines.For example to test if a vaccine composition assumes the sameconformation as a gp120 or gp41 peptide. Thus provided herein is amethod for detecting testing a vaccine, wherein the method includescontacting a sample containing the vaccine, such as a gp120 or gp41immunogen, with the antibody under conditions conducive to the formationof an immune complex, and detecting the immune complex, to detect thevaccine g in the sample. In one example, the detection of the immunecomplex in the sample indicates that vaccine component, such as such asa gp120 or gp41 immunogen assumes a conformation capable of binding theantibody.

In one embodiment, the antibody is directly labeled with a detectablelabel. In another embodiment, the antibody that binds gp120 or gp41 (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the antibody that binds gp120 or gp41 is utilized. As iswell known to one of skill in the art, a second antibody is chosen thatis able to specifically bind the specific species and class of the firstantibody. For example, if the first antibody is a human IgG, then thesecondary antibody may be an anti-human-IgG. Other molecules that canbind to antibodies include, without limitation, Protein A and Protein G,both of which are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

The immunoassays and method disclosed herein can be used for a number ofpurposes. Kits for detecting a polypeptide will typically comprise anantibody that binds gp120 or gp41, such as any of the antibodiesdisclosed herein. In some embodiments, an antibody fragment, such as anFv fragment or a Fab is included in the kit. In a further embodiment,the antibody is labeled (for example, with a fluorescent, radioactive,or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use. The instructional materials may be written, in anelectronic form (such as a computer diskette or compact disk) or may bevisual (such as video files). The kits may also include additionalcomponents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting gp120 or gp41 in a biological samplegenerally includes the steps of contacting the biological sample with anantibody which specifically reacts, under immunologically reactiveconditions, to gp120 or gp41. The antibody is allowed to specificallybind under immunologically reactive conditions to form an immunecomplex, and the presence of the immune complex (bound antibody) isdetected directly or indirectly.

F. Deposits

Plasmids including the nucleic acids encoding VRC01 heavy chain, VRC01light chain, VRC02 heavy chain, VRC02 light chain were deposited inaccordance with the Budapest Treaty at the American Type CultureCollection (ATCC) on Oct. 14, 2009. VRC01 heavy chain was deposited asATCC Deposit Number PTA-10412, VRC01 light chain was deposited as ATCCDeposit Number PTA-10411, VRC02 heavy chain was deposited as ATCCDeposit Number PTA-10414, and VRC02 light chain was deposited as ATCCDeposit Number PTA-10413. Plasmids including nucleic acid sequencesencoding the VRC03 heavy chain and VRC03 light chain were deposited inaccordance with the Budapest Treaty at the ATCC on Dec. 23, 2009. VRC03heavy chain was deposited as ATCC Deposit Number PTA-10551, VRC03 lightchain was deposited as ATCC Deposit Number PTA-10550.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Identification of Human Monoclonal HIV-1 Gp120Specific Neutralizing Antibodies

This example describes the isolation and characterization of the humanmonoclonal antibodies VRC01, VRC02, and VRC03.

As disclosed herein, using knowledge of Env structure together withcomputer-assisted protein recombinant forms of HIV-1 Env were designedthat specifically interact with neutralizing antibodies (Nabs) directedto the CD4 binding site (CD4bs) of the HIV gp120 glycoprotein. These Envprobes were used to identify and sort individual B cells expressingCD4bs antibodies, enabling the selective isolation of CD4bs-directedmonoclonal antibodies (mAbs) with extensive neutralization breadth. Togenerate a molecule that preserved the antigenic structure of theneutralizing surface of the CD4bs but eliminated other antigenic regionsof HIV-1, proteins were designed whose exposed surface residues weresubstituted with simian immunodeficiency virus (SIV) homologs and othernon-HIV-1 residues (FIG. 1A and FIG. 5). These changes were conferred ona core gp120 and a stabilized core gp120, both of which retained themajor contact surface for CD4 located on its outer domain. The gp120core lacked variable regions 1 to 3 and part of the amino and carboxytermini of the full gp120 molecule, and the stabilized core containedcrosslinks between different subregions of the core protein. Eightresurfaced proteins were designed and expressed, together with CD4bsmutants that served as negative controls by eliminating binding to theneutralizing mAb b12. Three resurfaced core Envs retained strongreactivity with b12 and mAb 2G12 (FIG. 6), the latter of whichrecognizes a surface glycan epitope and served as a positive control fora conformationally intact protein. The resurfaced stabilized core 3(RSC3) was chosen as the preferred candidate for further studies,because a greater percentage of its surface other than the outer domainCD4bs area was altered compared with the other variants (FIG. 6). Theconformational integrity and specificity of the RSC3 protein wasconfirmed by using a panel of known mAbs (FIG. 14, Table S1). Asexpected, RSC3 displayed strong reactivity to mAb b12 and little or noreactivity to a CD4 fusion protein (FIGS. 1B and 1C). RSC3 also reactedwith two weakly neutralizing CD4bs mAbs, b13 and m18, but it displayedno reactivity to four CD4bs mAbs that do not neutralize primary HIV-1isolates, nor with mAbs directed to other regions of the HIV-1 Env,including the coreceptor-binding region of gp120 and the V3 and CSregions of gp120 (FIG. 14, Table S1). DRSC3, which lacked a single aminoacid at position 371 that eliminated b12 binding, served as a negativecontrol. Together, these data confirmed the integrity of the antibodybinding surface of this resurfaced protein, and it was used for analysesof sera and to identify B cells from an HIV-1—infected individual whosesera contained broadly reactive NAbs.

A panel of broadly neutralizing sera was screened for the presence ofantibodies that could preferentially bind to RSC3 compared with DRSC3.CD4bs antibodies were detected in several sera, including serum fromdonor 45, which had been shown to contain NAbs directed to the CD4bs ofgp120 (FIG. 7). To determine whether antibodies that bind to RSC3 wereresponsible for the broad neutralization mediated by serum 45,neutralization studies were performed by using RSC3 to compete cognateantibodies. The utility of this assay was confirmed with the CD4bs mAbb12 and with mAb F105, which binds differently to the CD4bs and does notbind RSC3 (FIG. 14, Table S1). mAb b12 neutralizes many primary HIV-1strains, whereas F105 neutralizes mainly laboratory-adapted or otherhighly sensitive virus strains, such as the HXB2 strain. The addition ofRSC3 but not DRSC3 inhibited b12-mediated neutralization of HXB2. RSC3had no effect on F105 neutralization, and neither RSC3 nor DRSC3affected neutralization by the anti-V3 mAb 447-52D (FIG. 1D). Tointerrogate serum 45 neutralization, RSC3 competition studies wereperformed with this serum against a panel of diverseHIV-1 strains (FIG.1E). This analysis suggested that serum 45 neutralization wasprincipally directed against the CD4bs on functional viral spikes andthat the RSC3 faithfully mimicked this structure.

To isolate CD4bs-directed mAbs, a method of antigen-specific memory Bcell sorting was used together with single cell polymerase chainreaction (PCR), to amplify immunoglobulin G (IgG) heavy- and light-chaingenes from the cDNA of individual B cells. RSC3 and DRSC3 were expressedwith a tagged amino acid sequence that allows biotin labeling. The twoproteins could thus be distinguished by fluorescence-activated cellsorting (FACS) analysis after labeling with streptavidin (SA) conjugatedto the fluorochromes allophycocyanin (SA-APC) or phycoerythrin (SA-PE),respectively. Peripheral blood mononuclear cells (PBMC) from donor 45were incubated with RSC3 SA-APC and DRSC3 SA-PE, and singleantigen-specific memory B cells were sorted into wells of a microtiterplate after selecting for memory B cells (CD19+, CD20+, and IgG⁺) thatbound to the RSC3 but not the ΔRSC3 probe (FIG. 2A). Out of about 25million PBMC, 29 single RSC3-specific memory B cells were sorted, andthe matching heavy- and light-chain genes were successfully amplifiedfrom 12 cells. After cloning into IgG1 expression vectors thatreconstituted the heavy and light-chain constant regions, the full IgGmAbs were expressed. Three antibodies (VRC01, VRC02, and VRC03) boundstrongly to RSC3 and weakly or not at all to DRSC3 (FIG. 2B, left, FIG.2C, and FIGS. 8 and 9). To confirm the specificity of these antibodiesfor the CD4bs, enzyme linked immunosorbent assay (ELISA) was used totest the binding of each mAb against a wild-type gp120 and theCD4bs-defective Asp368→Arg368 (D368R) mutant (FIG. 2B, right). VRC01 andVRC02 bound with ≧100-fold lower relative affinity to the heavy chainvariable gene (VH) D368R mutant compared with wild-type gp120, and VRC03showed no detectable binding to the CD4bs knockout mutant. The ELISAbinding profile to an extended panel of mutant Env proteins furtherconfirmed the CD4bs specificity of VRC01, VRC02, and VRC03 (FIG. 14,Table 51).

Analysis of the heavy- and light-chain nucleotide sequences usingJoinSolver software (joinsolver.niaid.nih.gov) and the ImmunogeneticsInformation System (IMGT) database (imgt.cines.fr) revealed that VRC01and VRC02 were somatic variants of the same IgG1 clone. The heavy-chainCDR3 region of both mAbs was composed of the same 14 amino acids (FIG.10), and both mAbs were highly somatically mutated, with 32% of theheavy chain variable gene (VH) and 17 to 19% of the kappa light chainvariable gene (VK) nucleotides divergent from putative germline genesequences. VRC03 was potentially derived from a different IgG1 clone,but its heavy chain was derived from the same IGHV1-02*02 and IGHJ1*01alleles as VRC01 and VRC02. VRC03 was also highly somatically mutated,with an unusual seven-amino acid insertion in heavy-chain framework 3and 30% of VH and 20% of VK nucleotides divergent from putative germlinegene sequences. The heavy chain CDR3 of VRC03 contained 16 amino acids.All three mAbs share common sequence motifs in heavy-chain CDR1, CDR2,and CDR3.

The binding characteristics of the mAbs were further analyzed by surfaceplasmon resonance (SPR), competition ELISA, and isothermal titrationcalorimetry (ITC). SPR demonstrated that VRC01 (KD=3.88×10-9M) and VRC02(KD=1.11×10-8 M) bound gp120 with high affinity whereas VRC03 reactedwith about 10-fold lower affinity (KD=7.31×10-8 M). To evaluate theepitope reactivity of these mAbs on gp120, competition ELISAs wasperformed with a panel of well-characterized mAbs. As expected, bindingby all three VRC mAbs was competed by CD4bs mAbs b12 and F105 and byCD4-Ig (FIG. 3A left and FIG. 11A). Unexpectedly, the binding of mAb 17bto its site in the coreceptor binding region of gp120 was markedlyenhanced by the addition of VRC01 or VRC02 (FIG. 3A right). Thisenhancing effect was similar, although not as profound, as the knowneffect of CD4-Ig. In contrast, mAb b12 inhibited mAb 17b binding (FIG.3A right), as previously shown. A similar enhancing result was observedfor VRC01 in an assay that measures gp120 binding to its CCRS coreceptor(FIG. 11C). Thus, VRC01 and VRC02 act as partial CD4 agonists in theirinteraction with gp120, whereas VRC03 does not display this effect.Thermodynamic analysis by ITC provided data consistent with the ELISAresults and demonstrated a change in enthalpy (-DH) associated with theVRC01-gp120 interaction that was similar to the interaction of CD4-Igand gp120 (FIG. 3B), further demonstrating that VRC01 binding inducedconformational changes in gp120. In contrast to the data for gp120binding, VRC01 did not enhance viral neutralization by mAb 17b (FIG.12). These data suggest that VRC01 and VRC02 partially mimic theinteraction of CD4 with gp120. This may explain their broad reactivity,because essentially all HIV-1 isolates must engage CD4 for cell entry.

The potency and breadth of neutralization by VRC01, VRC02, and VRC03,compared with those by b12 and CD4-Ig, were assessed on a comprehensivepanel of Env pseudoviruses (FIGS. 4 and 15-24, Table S2a-S2j). These 190viral strains represented all major circulating HIV-1 genetic subtypes(clades) and included viruses derived from acute and chronic stages ofHIV-1 infection. VRC01 neutralized 91% of these viruses with a geometricmean value of 0.33 mg/ml (FIGS. 4 and 15-24, Table S2a-S2j). The datafor VRC02 were very similar (FIGS. 15-24, Table S2a-S2j). Of note, thesemAbs were derived from an HIV-1 clade B—infected donor yet displayedneutralization activity against all genetic subtypes of HIV-1. VRC03 wasless broad than VRC01 and VRC02, neutralizing 57% of the viruses (FIGS.15-24, Table S2a-S2j). In contrast, b12, also derived from a cladeB-infected donor, neutralized 41% of viruses tested. Because VRC01 wasderived from a donor whose sera was also broadly neutralizing, therelationship between the neutralization breadth and potency of serum 45IgG and mAb VRC01 was also assessed. Among 140 viruses tested, there wasa significant association (P=0.005; Fisher's exact test) between thenumber of viruses neutralized by serum 45 IgG and the number neutralizedby VRC01 (FIG. 13A). Among the 122 viruses neutralized by both serum IgGand VRC01, there was a strong association (P<0.0001; Deming linearregression) between the neutralization potency of the serum IgG and thepotency of VRC01 (FIG. 13B). Therefore, although VRC01 did not accountfor all serum 45 IgG neutralization, the VRC01-like antibody specificitylargely accounts for the extensive breadth and potency of serum 45.These findings demonstrate that a focused B cell response can target ahighly conserved region of the HIV-1 Env in humans.

Other mAbs are able to neutralize HIV-1, but none has a profile ofpotency and breadth similar to VRC01 and VRC02. Antibody 4E10 requiresrelatively high concentrations to neutralize primary strains of HIV-1,and it neutralizes only 12% of Env-pseudoviruses at a concentration ofless than 1 μg/ml. The well-characterized CD4bs mAb b12 and the morerecently described HJ16 are informative with respect to antigenrecognition, but each display restricted breadth (˜40% of HIV-1strains). Recently, two broadly neutralizing somatic variant mAbs, PG16and PG9, were isolated by high-throughput neutralization screening of Bcell supernatants. The PG16 and PG9 neutralized 73% and 79%,respectively, of viruses tested and recognized a glycosylated region ofHIV-1 Env that is present on the native viral trimer, but this epitopeis not well presented on gp120 or gp140. VRC01 and VRC02 access theCD4bs region of gp120 in a manner that partially mimics the interactionof CD4 with gp120. This observation may explain their impressive breadthof reactivity. The isolation of these mAbs from an HIV-1—infected donorand the demonstration that they neutralize the vast majority of HIV-1strains by targeting the functionally conserved receptor binding regionof Env provides proof of concept that such antibodies can be elicited inhumans. The discovery of these mAbs provides new insights into how thehuman immune system is able to effectively target a vulnerable site onthe viral Env.

Materials and Methods

Human Specimens.

The sera and peripheral blood mononuclear cells (PBMC) described in thisExample were from HIV-1 infected individuals enrolled in investigationalreview board approved clinical protocols at the National Institute ofAllergy and Infectious Diseases. Donor 45, from whom mAbs VRC01, VRC02and VRC03 were isolated, has been HIV-1 infected with a clade B virusfor more than 15 years. He is a slow progressor with CD4 T-cell countsover 500 cells/μl, plasma HIV-1 RNA values less than 15,000 copies/ml.He has not initiated antiretroviral treatment.

Computational Design of the Antigenically Resurfaced Core (RC) andResurfaced Stabilized Core (RSC) Proteins.

The atomic level structures of HIV-1 gp120 in complex with CD4 (ProteinData Bank (PDB) ID: 2NXY), b12 (PDB ID: 2NY7), and F105 (PDB ID: 3HI1)defined the CD4-binding footprint, and neutralizing (b12) as well asnon-neutralizing (F105) antibody epitopes on gp120. These structureswere used to guide the computational design of new gp120 proteins thatmaintain the b12 neutralizing epitope but modify the antigenic surfaceoutside the b12 epitope. Modifications outside the b12 epitope included,but were not limited to, mutations to eliminate CD4 and F105 binding andtrimming the V1/V2 to eliminate co-receptor epitopes. Designs of most ofthe resurfaced proteins were based on the wild-type HXB2 core in PDB ID:2NXY to optimize expression and folding. However, since the stabilizedcore version of gp120 HXB2 Ds12F123 eliminates binding to mostnon-neutralizing antibodies and keeps b12 binding intact, some designs(including RSC3) were based on the stabilized core version of gp120 HXB2Ds12F123.

The general algorithm of the resurfacing design is illustrated in FIG.5A. First, candidate resurfacing positions on gp120 were identified assurface exposed positions that do not contact the antibody (b12) and arenot within or near an N-glycosylation site. Next, the set of amino acidsallowed at each resurfacing position was assigned semi-automatically,employing a combination of different types of information (evolutionaryinformation, structural and 2 solubility considerations, andsimilarity/differences with wild-type or pre-existing designs). Finally,RosettaDesign was used to select low energy sequences. Different finaldesigns were generated largely by devising different sets of allowedamino acids at each design position, but also by modifying the designpositions themselves. The genes of the resurfaced proteins weresynthesized for cloning, and the RC and RSC proteins were expressed andcharacterized for antigenic properties. What follows are details on theprocess and the individual designs.

Identification of Resurfacing Positions.

CD4- and b12-contacting residues as well as the surface accessibility ofeach residue on gp120 were determined based on the gp120-CD4 andgp120-b12 structures (PDB ID 2NXY and 2NY7). Surface exposed residueswere defined using the program NACCESS (bioinf.manchester.ac.uk/naccess)as residues with >40% side-chain surface area exposed, relative to thesame side-chain in an isolated tripeptide. Antibody contact residueswere defined as any gp120 residue with at least one heavy atom within8.0 angstroms of a heavy atom on the antibody. Residues nearN-glycosylation sites were defined as any residue with at least oneside-chain heavy atom within 6.0 angstroms of any heavy atom on eitherthe Nacetyl-glucosamine (NAG) group or the asparagine of aN-glycosylation site (NXS/T, where X is any residues except proline).Initially, 49 candidate positions were identified on b12-bound gp120(PDB ID: 2NY7), but the above criteria were relaxed in some cases toallow additional design positions, and in other cases design positionswere restricted to generate pairs of molecules with resurfacingmutations at complementary sets of positions.

Semi-Automatic Assignment of Amino Acid Libraries.

Different strategies were used to assign libraries of allowed aminoacids at each resurfacing position, in order to obtain different finalsequences from RosettaDesign. For the design of RSC2, amino acids from amultiple sequence alignment of HIV-1 HXB2 with SIV (hiv.lanl.gov) wereallowed (evolutionary information), but most hydrophobic residues weredisallowed unless packed on the surface of a beta sheet, and all polarand the native HIV-1 residues were allowed (structural and solubilityconsiderations). RC1 was derived from RSC2 by threading the final RSC2sequence onto the CD4-bound structure (2NXY) and reverting mutationsthat would destabilize the CD4-bound conformation. The design of RSC3was carried out following experimental feedback that RSC2 successfullymaintained nM b12 affinity. For RSC3, most mutations from RSC2 wereenforced, a wider range of amino acids were allowed at some positionsthat had not been mutated in RSC2, and additional resurfacing positionswere selected based on both exposure and distance from mutations inRSC2. The goal was to ensure that as many potential antibody footprintsof area ˜20 Å2 outside the b12-binding site as possible contained atleast one mutation. The criteria for resurfacing positions were relaxedfor RSC3—eight of the new positions were near a NAG, and six wereslightly less than 40% exposed. Finally, the native amino acid was notallowed at the new RSC3 design positions, guaranteeing increasedresurfacing surface coverage. Resurfacing positions and allowed aminoacids for RC4-8 were designed to increase the resurfaced area andantigenic diversity of RC1, following experimental feedback that RC1maintained high b12 affinity. RC8 was generated using an expanded set ofdesign positions and nearly only polars were allowed at all designpositions. RC7 was generated using the same design positions as RC8, butthe amino acids chosen for RC8 were disallowed at most design positions,and native amino acids were disfavored directly by assigning them asmall energetic penalty. RC4 and RC5 utilized different resurfacingpositions compared to RC1, RC7, and RC8 wherever possible, wererestricted to polar mutations, and RC5 was designed to be antigenicallydifferent than RC4 by disallowing amino acids chosen for RC4. RC6 usedthe same design positions as RC1, but expanded beyond those positions,and disallowed the amino acids used in RC1.

Rosettadesign Parameters.

In all cases non-exposed amino acids were held fixed at the nativerotamer. In most cases surface exposed amino acids that were kept asnative were also held fixed at the native rotamer. For design of RSC3,amino acids designed into the parent RSC2 were allowed to repack duringdesign of RSC3. The lowest energy design for a particular combination ofresurfacing positions and allowed amino acids was selected forexperimental testing.

RSC2 and RSC3 Sequences.

The final RSC2 and RSC3 designs contained 34 and 61 mutations relativeto the stabilized core (HXB2 Ds12F123) in PDB ID: 2NY7, respectively(not including the V1/V2 trim discussed below). The stabilized core hasa total of 330 amino acids, so the resurfacing mutations in RSC2 andRSC3 amounted to modifications of 10% and 18% of the protein,respectively. For RSC3, 82% (50/61) of the mutations were contained inthe SIV multiple sequence alignment and 18% (11/61) were not,illustrating that structural and solubility considerations contributedto the design. Only 11 (18%) of the final RSC3 mutations were 4contained within the sequence of SIVmac32H, illustrating the importanceof using a multiple sequence alignment rather than a single sequence.Mutations for all the resurfaced proteins are highlighted in thesequence alignment in FIG. 5B.

Eliminating CD4 and F105 Binding.

RSC2 and RSC3 were designed on the stabilized core that alreadyeliminates F105 binding. To eliminate CD4 binding, the resurfacingdescribed above was expected to suffice due to mutations of two CD4contact residues in the β20/21 (mutations were N425G and W427V) and dueto the favored b12-bound conformation of the β20/21 over the bridgingsheet conformation necessary for CD4 binding (by mutations in β20/21 andin α1 which is linked to β2021 by a disulfide in RSC3). To eliminateF105-binding for RC1 and RC4-8 mutations at key F105 contact locationswere added to the resurfacing design. To eliminate CD4 binding for RC1and RC4-8, the β20/21 was truncated to GG between I423 and Y435.

V1/V2 Stem Trimming.

The V1/V2 was trimmed differently in different constructs, attempting tofind minimal truncation while maintaining high protein expression. RSC2utilized the same V1N2 trim as the stabilized core in the b12-crystalstructure (PDB ID: 2NY7), but RSC3 had a more aggressively truncatedV1/V2 (see alignment in FIG. 5B). RC1 and RC4-8 used a V1/V2 trimsequence of VKLTPLAGATSVITQA (SEQ ID NO. 1466) between C119 and C205.

Protein Expression and Purification.

Genes for HXB2 core, the stabilized core (HXB2 Ds12F123) and thedesigned RC and RSCs were each synthesized with a C-terminal His tag byGeneArt (Regensburg, Germany), and cloned into a mamalian CMV/Rexpression vector. Proteins were produced by transient tranfection using293fectin (Invitrogen, Carlsbad, Calif.) in 293F cells, a humanembryonic kidney cell line (Invitrogen) maintained in serum-freefree-style medium (Invitrogen). Culture supernatants were harvested 4-5days after transfection, filtered through a 0.45 μm filter, andconcentrated with buffer-exchange into 500 mM NaCl, 50 mM Tris (pH 8.0).Proteins were first purified by Co-NTA (cobalt-nitrilotriacetic acid)chromatography method using a HiTrap IMAC HP column (GE Healthcare,Piscataway, N.J.). The peak fractions were collected, and furtherpurified by gel-filtration using a HiLoad 16/60 Superdex 200 pg column(GE Healthcare). The fractions containing monomers of each protein werecombined, concentrated and flash frozen at −80° C. To generate thesurface plasmon resonance (SPR) data 5 shown in FIG. 2C and FIGS. 8 and9, both RSC3 and the stabilized core (Ds12F123) were further purifiedusing b12 affinity column. Monomeric gp120s were expressed by transienttransfection of 293F cells.

Surface Plasmon Resonance (SPR).

The binding kinetics of gp120 variants with different ligands wereassessed by SPR analysis on Biacore 3000 or Biacore T-100 (GEHealthcare). HIV6 1 gp120-reactive ligands (CD4-Ig and specific mAbs)were either immobilized directly onto a CMS sensor chip with standardamine coupling or captured with a mouse anti-human IgG Fc antibodysupplied in the “human antibody capture kit” (GE Healthcare) to asurface density about 300 response units (RU). Variant gp120s at 5-200nM were first passed over the modified sensor chips at 30 μl/min for 3-4minutes, followed by a 5 minutes dissociation phase to identify roughbinding affinities. Then a 2-fold increasing series of gp120concentrations were passed over the chip, with the concentration of theseries adjusted so that at least three runs resulted in maximum 10-150RU.

Accompanying each gp120 series, blank reference using buffer to mockgp120 was included. The buffer in all studies was 10 mM HEPES, pH 7.4,150 mM NaCl, 3 mM EDTA, and 0.01% surfactant P-20. Sensorgrams werecorrected with blank reference and fit globally with Biacore Evaluationsoftware using a 1:1 Langmuir model of binding. Although CD4-Ig andother ligands might formally be analyzed with a two-state binding model,such treatment should not affect the primary on-rates nor overall KDsreported here.

ELISA analyses. Each antigen in PBS (pH 7.4) at 2 μg/ml was used to coatplates overnight at 4° C. Coated plates were blocked with B3T buffer(150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 3.3% fetal bovine serum, 2%bovine albumin, 0.07% Tween 20) for 1 hour at 37° C., followed byincubation with sera or antibody serially diluted in B3T buffer for 1hour at 37° C. Horseradish peroxidase (HRP)-conjugated goat anti-humanIgG Fc antibody (Jackson ImmunoResearch Laboratories Inc., West Grove,Pa.) at 1:10,000 was added for 1 hour at 37° C. All volumes were 100μl/well except that 200 μl/well was used for blocking. Plates werewashed between each step with 0.1% Tween 20 in PBS. Plates weredeveloped using either 3,3′,5,5′-tetramethylbenzidine (TMB) (Kirkegaard& Perry Laboratories) and read at 450 nm, or ophenylenediaminedihydrochloride (Sigma) and read at 490 nm. For competitive ELISAanalyses, plates were coated with 1 μg/ml of a sheep anti-gp120 C5antibody, D7324 (Cliniqa Corp., Fallbrook, Calif.) or 10 μg/ml ofGalanthus nivalis lectin (Sigma) to capture 2 μg/ml of purified YU2gp120 or RSC3 respectively. After blocking, serial dilutions of thecompetitor antibodies or CD4-Ig were added to the captured gp120 or RSC3in 50 μl of B3T buffer, followed by adding 50 μl of biotin-labeledantibody at fixed concentrations: 100 ng/ml for VRC01 and VRC02 for bothproteins, 4 μg/ml for VRC03 for YU2 gp120 or 100 ng/ml for RSC3, and 20ng/ml for 17b for YU2 gp120. The plates were incubated at 37° C. for 1hour, followed by incubation with 250 7 ng/ml of streptavidin-HRP(Sigma) at ambient temperature for 30 minutes, and developed with TMB asdescribed.

Isothermal Titration Calorimetry (ITC).

Isothermal titration calorimetry (ITC) was carried out using ITC200microcalorimeter system from MicroCal, Inc. All proteins were thoroughlydialyzed against PBS before use. The dialysis buffer was filteredthrough a 0.2 μm membrane and used to dilute the protein samples. Theconcentration of gp120 in the sample cell was approximately 5 μM andthat of CD4-Ig or mAbs in the syringe was approximately 25 μM. The molarconcentrations of the proteins were calculated using the following molarextinction coefficients: gp120, 1.52; CD4-Ig, 1.2; b12, 1.46; VRC01,1.53; VRC03, 1.57. Gp120 in the sample cell were titrated to saturationby the stepwise addition of 2 μl of ligand from the syringe at 120-secintervals at 37° C. The heat evolved upon each injection was obtainedfrom the integral of the calorimetric signal. The values for enthalpy(ΔH) and entropy (ΔS) were obtained by fitting the data to a nonlinearleast-squares analysis with Origin software.

Viral Entry, Neutralization and Protein Competition Assays.

Neutralization was measured using single round infection by HIV-1Env-pseudoviruses and TZM-bl target cells. Neutralization curves werefit by nonlinear regression using a 5-parameter hill slope equation. The50% and 80% inhibitory concentrations (IC50 and IC80) were reported asthe antibody concentrations required to inhibit infection by 50% and 80%respectively. Competition of serum or mAb neutralization was assessed byadding a fixed concentration (25 μg/ml) of the RSC3 or ΔRSC3glycoprotein to serial dilutions of antibody for 15 min prior to theaddition of virus. The resulting IC50 values were compared to thecontrol with mock protein added. The neutralization blocking effect ofthe proteins was calculated as the percent reduction in the IC50 valueof the antibody in the presence of protein compared to PBS. Synergisticor additive neutralization was assessed by mixing a fixed concentration(10 μg/ml) of the test antibody with serial dilutions of sCD4, CD4-Ig orVRC01 for 15 min prior to the addition of virus. The baseline of viralentry at each concentration of sCD4, CD4-Ig or VRC01 was used tocalculate the adjusted percent neutralization. Neutralization was alsoassessed 8 using Env-pseudoviruses generated by 293T transfection usingthe pNL4-3 ΔEnv HIV-1 backbone containing a luciferase reporter gene toinfect activated PBMC. Neutralizations using uncloned PBMC-derived HIV-1primary isolates were performed by single-round infection of eitherTZM-bl cells using luciferase as readout, or activated PBMC using flowcytometry staining for HIV-1 p24 antigen. CD4-facilitated virus entrywas performed in the CCR5+/CD4− cell line Cf2Th/syn CCR5 withEnv-pseudoviruses containing the luciferase pNL4-3 ΔEnv HIV-1 backbone.A mixture of 40 μl of viral stock and 10 μl of serial dilutions of sCD4,CD4-Ig or VRC01 was incubated at 37° C. for 30 min before adding 1×104Cf2Th/syn CCR5 cells. Virus entry was measured 2 days later byluciferase activity in cell lysates.

Construction of the HIV-1 Envelope Sequence Dendrogram.

HIV-1 gp160 protein sequences (excluding the signal peptide) of HXB2 andthe 190 isolates used in the neutralization assays were aligned usingMUSCLE, for multiple sequence comparison by log-expectation. The proteindistance matrix was calculated by “protdist” and the dendrogram wasconstructed using the neighbor joining method by “Neighbor”. Allanalysis and the programs used were performed at the NIAID Biocluster(niaid-biocluster.niaid.nih.gov). The tree was displayed withDendroscope. Isolation of antigen-specific memory B cells byfluorescence activated cell sorting (FACS). The plasmid constructs forRSC3 and ΔRSC3 were modified by the addition of the sequence encodingthe Avi-tag signal for biotinylation (LNDIFEAQKIEWHE, SEQ ID NO: 26) atthe 3′ end of the gene, and the modified genes were subcloned into theCMV/R expression vector. After expression and purification, the proteinswere biotinylated at 40 μM utilizing biotin ligase Bir A (Avidity,Denver, Colo.) at 30° C. for 30 min, followed by removal of excess freebiotin and buffer exchange with PBS (pH 7.4) using a 30-kDa Centriconplus filter (Millipore). Biotinylation of the RSC proteins was confirmedby ELISA. To conjugate proteins with the streptavidin-fluorochromereagents, in a stepwise process, 1/5 of the molar equivalent of thestreptavidin-fluorochrome reagent was added to the biotinylated RSC3 orΔRSC3 at 20-min intervals until the molar ratio ofstreptavidin-fluorochrome reagent: biotinylated protein reached 1:1. Theincubation was carried out at 4° C. with gentle rocking.Streptavidin-allophycocyanin (SA-APC) (Invitrogen) was 9 mixed withbiotinylated RSC3 and streptavidin-phycoerythrin (SA-PE) (Sigma) wasmixed with biotinylated ΔRSC3. Thus, each protein carried a differentfluorochrome: RSC3—SA-APC and ΔRSC3—SA-PE.

Antigen specific B cells were identified with a panel of ligandsincluding fluorescently labeled antibodies for CD3, CD8, CD19, CD20,CD27, CD14, IgG and IgM. PBMC were stained with an antibody cocktailconsisting of anti-CD3-APC-Cy7 (BD Pharmingen), CD8-Qdot705 (VRC),CD19-Qdot585 (VRC), CD20-Pacific Blue (VRC), CD27-APC-AlexaFluor700(Beckman Coulter), CD14-Qdot800 (VRC), IgG-FITC (BD Pharmingen), andIgM-PE-Cy5 (BD Pharmingen). In addition, aqua blue (Invitrogen) was usedto exclude dead cells. A total of 25 million cryopreserved PBMC werethawed and resuspended in 10 ml RPMI 1640 medium (Invitrogen) with 10%fetal bovine serum pre-warmed to 37° C. and treated with 20 μg/ml DNaseI (New England Biolabs, Ipswich, Mass.), followed by centrifugation at860×G for 5 min. Medium was removed and the cells were resuspended in 10ml chilled PBS followed by centrifugation at 860 ×G for 5 min. The cellpellet was resuspended in 50 μl of chilled PBS with the aqua blue dyeand stained at 4° C. in dark for 20 min. The antibody cocktail and theRSC3 and ΔRSC3 multimers, in a total volume of 50 μl, was added to thecells and incubated at 4° C. in dark for 1 hour. The cells were washedwith 10 ml cold PBS, resuspended in 2 ml cold PBS and passed through a70 μm cell mesh (BD Biosciences). The stained PBMC were analyzed andsorted using a modified 3-laser FACSAria cell sorter using the FACSDivasoftware (BD Biosciences). Fluorescence compensation was performed usinganti-mouse Ig Kappa compensation beads (BD Biosciences) stained witheach antibody in a separate tube. For the CD3-APC-Cy7 antibody,anti-mouse IgH&L COMPtrol beads (Spherotech, Lake Forrest, IL) were usedand the aqua blue fluorescence was compensated using pre-labeledamine-beads. Single cells with the phenotype of CD3−, CD8−, aqua blue-,CD14−, CD19+, CD20+, IgG+, IgM−, RSC3+ and ΔRSC3− were defined as CD4bsdirected antigen specific B cells, and single cells were sorted into96-well PCR plates containing 20 μl of lysis buffer per well. The lysisbuffer contained 0.5 μl of RNase Out (Invitrogen), 5 μl of 5× firststrand buffer (Invitrogen), 1.25 μl of 0.1M DTT (Invitrogen) and 0.0625μl of Igepal (Sigma). The PCR plates with sorted cells were quicklyfrozen on dry-ice and stored at −80° C. The total content of the patientPBMC sample 10 passing through the sorter was saved in FCS files forfurther analysis with FlowJo software (TreeStar, Cupertino, Calif.).

Single B-Cell RT-PCR and Subsequent Sequencing and Cloning.

For each sorted cell, the IgG heavy and the corresponding Ig light chaingene transcripts were amplified by RT-PCR and cloned into eukaryoticexpression vectors to produce full IgG1 antibodies. The frozen plateswith single B-cell RNA were thawed at room temperature, and the RTreaction was carried out by adding 3 μl of random hexamers at 150 ng/μ,2 μl of dNTP mix, each at 10 mM, and 1 μl of SuperScript III(Invitrogen) into each well. The thermocycle program for RT was 42° C.for 10 min, 25° C. for 10 min, 50° C. for 60 min and 94° C. for 5 min.The cDNA plates were stored at −20° C., and the IgH, Igκ and Igλvariable region genes were amplified independently by nested PCRstarting from 5 μl of cDNA as template. All PCRs were performed in96-well PCR plates in a total volume of 50 μl containing water, 5 μl of10× buffer, 1 μl of dNTP mix, each at 10 mM, 1 μl of MgC12 at 25 mM(Qiagen) for 1st round PCR or 10 μl 5× Q-Solution (Qiagen) for 2nd roundPCR, 1 μl of primer or primer mix for each direction at 25 μM, and 0.4μl of HotStar Taq DNA polymerase (Qiagen). Each round of PCR wasinitiated at 94° C. for 5 min, followed by 50 cycles of 94° C. for 30sec, 58° C. for IgH and Igκ or 60° C. for Igλ for 30 sec, and 72° C. for1 min, followed by 72° C. for 10 min. The positive 2nd round PCRproducts were cherry-picked for direct sequencing with both forward andreverse PCR primers. PCR products that gave a productive IgH, Igκ or Igλrearranged sequence were re-amplified from the 1st round PCR usingcustom primers containing unique restriction digest sites andsubsequently cloned into the corresponding Igyl, Igκ and Igλ expressionvectors. The full-length IgG1 was expressed by cotransfection of 293Fcells with equal amounts of the paired heavy and light chain plasmids,and purified using a recombinant protein-A column (GE Healthcare).

Igg Gene Family Analysis.

The IgG heavy and light chain nucleotide sequences of the variableregion were analyzed with JoinSolver (Joinsolver.niaid.nih.gov) andusing the IMGT database (imgt.cines.fr). Normal donor peripheral blooddata originated from 120 IgD+CD27+ and 97 IgD-CD27+ sequences pooled forheavy chain analysis and 167 mutated IgM+ sequences for kappa chainanalysis. The VRC mAb VK gene use was 11 determined by homology togermline genes in the major 2p11.2 IGK locus. VRC mAb D gene use wasdetermined by homology to genes in the major 14q32.33 IGH locus.

Env and CCR5 Cell Surface Staining.

293T cells were transfected with plasmid DNA encoding JRFL Env toexpress the envelope glycoprotein on the cell surface. Cells werestained with anti-Env mAbs. The FACS signal was generated by adding asecondary antibody, goat anti-human IgG F(ab′)2 conjugated withphycoerythrin (SouthernBiotech), at 1:125. Data were collected usingflow cytometry with the BD LSR Flow Cytometer, and binding curves weregenerated by plotting the mean fluorescence intensity (MFI) as afunction of antibody concentration. To assess gp120 binding to CCR5 onthe surface of cells, biotinylated gp120 was used at 5 μg/ml to stainthe CCR5 expressing canine thymus cell line, Cf2Th/syn CCR5. Prior tothe staining of Cf2Th/syn CCR5 cells, biotinylated gp120 was incubatedwith ligands including CD4-Ig, VRC01, VRC02, VRC03 and b12 at serialconcentrations ranging from 0.04-25 μg/ml. A streptavidin-APC conjugate(Invitrogen) was used at 1 μg/ml to stain Cf2Th/syn CCR5 cells togenerate FACS signal, and binding data were collected using flowcytometry with the BD LSR Flow Cytometer. All the staining andincubations were carried out at room temperature for 1 hour.

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism version 5.0(GraphPad Software Inc.). A two-sided Fisher's Exact Test at alpha=0.05was used for assessing the relationship between the viral sensitivity toserum 45 IgG and to VRC01. Among the viruses that were sensitive toboth, Deming Regression was used to model the relationship on the log 10scale, allowing for measurement error in the IC50s for both the serumIgG and the mAb. These models were run under the assumption of equalerror variance. As a sensitivity analysis, the regression models wererebuilt with an estimated variance ratio; although the slope estimatechanged slightly, the conclusions were consistent.

The amino acid sequences of the heavy and light chains of VRC01, VRC02and VRC03 are shown below (SEQ ID NOs: 1-4, 27 and 28). Bold residuesindicate the locations of the CDRs. Exemplary nucleic acid sequencesencoding the heavy and light chains of VRC01, VRC02 and VRC03 (SEQ IDNOs: 29-34) are also shown below.

VRC01 Heavy Chain (SEQ ID NO: 1):QVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSPVRC01 Light Chain (SEQ ID NO: 2):EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGDFGVYYCQQYEFFGQGTKVQVDIKR VRC02 Heavy Chain (SEQ ID NO: 3):QVQLVQSGGQMKKPGESMRISCQASGYEFIDCTLNWVRLAPGRRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTADDTAVYYCTRGKNCDYNWDFEHWGRGTPVTVSSAVRC02 Light Chain (SEQ ID NO: 4):EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLTIRNLESGDFGLYYCQQYEFFGQGTKVQVDIKR VRC03 Heavy Chain (SEQ ID NO: 27):QVQLVQSGAVIKTPGSSVKISCRASGYNFRDYSIHWVRLIPDKGFEWIGWIKPLWGAVSYARQLQGRVSMTRQLSQDPDDPDWGVAYMEFSGLTS/PADTAEYFCVRRGSCDYCGDFPWQYWGQGTWWSS AVRC03 Light Chain (SEQ ID NO: 28):EIVLTQSPGILSLSPGETATLFCKASQGGNAMTWYQKRRGQVPRLLIYDTSRRASGVPDRFVGSGSGTDFFLTINKLDREDFAVYYCQQFEFFGLGSELEVH VRC01 Heavy Chain (SEQ ID NO: 29):CAGGTGCAGCTGGTGCAGTCTGGGGGTCAGATGAAGAAGCCTGGCGAGTCGATGAGAATTTCTTGTCGGGCTTCTGGATATGAATTTATTGATTGTACGCTAAATTGGATTCGTCTGGCCCCCGGAAAAAGGCCTGAGTGGATGGGATGGCTGAAGCCTCGGGGGGGGGCCGTCAACTACGCACGTCCACTTCAGGGCAGAGTGACCATGACTCGAGACGTTTATTCCGACACAGCCTTTTTGGAGCTGCGCTCGTTGACAGTAGACGACACGGCCGTCTACTTTTGTACTAGGGGAAAAAACTGTGATTACAATTGGGACTTCGAACACTGGGGCCGGGGCACCCCGGTCATCGTCTCATCACCVRC01 Light Chain (SEQ ID NO: 30):GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAACAGCCATCATCTCTTGTCGGACCAGTCAGTATGGTTCCTTAGCCTGGTATCAACAGAGGCCCGGCCAGGCCCCCAGGCTCGTCATCTATTCGGGCTCTACTCGGGCCGCTGGCATCCCAGACAGGTTCAGCGGCAGTCGGTGGGGGCCAGACTACAATCTCACCATCAGCAACCTGGAGTCGGGAGATTTTGGTGTTTATTATTGCCAGCAGTATGAATTTTTTGGCCAGGGGACCAAGGTCCAGGTCGACATTAAGCGAVRC02 Heavy Chain (SEQ ID NO: 31):CAGGTGCAGCTGGTGCAGTCTGGGGGCCAGATGAAGAAGCCTGGCGAGTCGATGAGAATTTCTTGTCAGGCTTCCGGATATGAATTTATTGATTGTACACTAAATTGGGTTCGCCTGGCCCCCGGAAGAAGGCCTGAATGGATGGGATGGCTGAAGCCTCGAGGGGGGGCCGTCAACTACGCACGTCCACTTCAAGGCAGAGTGACCATGACTCGAGACGTGTATTCCGACACAGCCTTTTTGGAGCTGCGCTCCTTGACAGCAGACGACACGGCCGTCTACTATTGTACTAGGGGAAAAAATTGTGATTACAATTGGGACTTCGAACACTGGGGCCGGGGTACCCCGGTCACCGTCTCATCAGCCVRC02 Light Chain (SEQ ID NO: 32):GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAACAGCCATCATCTCTTGTCGGACCAGTCAGTATGGTTCCTTAGCCTGGTATCAACAGAGGCCCGGCCAGGCCCCCCGGCTCGTCATCTATTCGGGCTCTACTCGGGCCGCAGGCATCCCAGACAGGTTCAGCGGCAGTCGGTGGGGGCCAGACTACAATCTCACCATCAGGAACCTGGAGTCGGGAGATTTTGGTCTTTATTATTGCCAGCAGTATGAATTTTTTGGCCAGGGGACCAAGGTCCAGGTCGACATTAAGCGAVRC03 Heavy Chain (SEQ ID NO: 33):CAGGTGCAGCTGGTGCAGTCTGGGGCTGTGATTAAGACGCCTGGGTCCTCAGTGAAGATCTCATGTCGGGCTTCTGGATACAACTTTCGTGATTATTCGATCCATTGGGTCCGCCTCATTCCTGACAAGGGATTTGAGTGGATTGGATGGATTAAACCTCTGTGGGGTGCCGTCAGTTATGCCCGGCAACTTCAGGGCCGAGTCTCTATGACTCGACAATTATCTCAAGACCCAGACGACCCGGACTGGGGCGTTGCCTACATGGAGTTCAGTGGACTGACGT/CCCGCCGACACGGCCGAATATTTTTGTGTCCGGAGAGGGTCCTGTGATTATTGCGGAGACTTTCCCTGGCAATACTGGGGTCAGGGCACCGTCGTCGTCGTCTCGTCAGCG VRC03 Light Chain (SEQ ID NO: 34):GAAATTGTGTTGACGCAGTCTCCCGGCATCCTGTCTCTGTCTCCAGGAGAGACAGCCACCCTCTTTTGTAAGGCCAGTCAGGGTGGCAATGCTATGACGTGGTATCAGAAGAGACGTGGCCAGGTTCCCAGACTCCTGATCTACGATACATCTCGCAGGGCCTCTGGCGTTCCTGACAGATTTGTTGGCAGTGGGTCTGGGACAGACTTCTTTCTCACGATCAACAAATTGGACCGGGAAGATTTCGCAGTCTATTATTGTCAACAATTTGAATTTTTTGGCCTGGGGAGCGAGCTGGAAGTCCATCGA

Example 2 Characterization of the Atomic Structure of VRC01 and gp120

This example describes the crystal structure for one of the VRC01antibody in complex with an HIV-1 gp120 core. As disclosed herein themolecular basis of VRC01 neutralization is deciphered. In addition themechanisms of natural resistance of HIV are identified and it shown howVRC01 minimizes such resistance.

To gain a structural understanding of VRC01 neutralization theantigen-binding fragment (Fab) of VRC01 was crystallize in complex withan HIV-1 gp120 from the clade A/E recombinant 93TH057. The crystallizedgp120 consisted of its inner domain—outer domain core, with truncationsin the variable loops V1/V2 and V3 as well as theN- and C-termini, whichare regions known to extend away from the main body of the gp120envelope glycoprotein. Diffraction to 2.9 Å resolution was obtained fromorthorhombic crystals, which contained four copies of the VRC01-gp120complex per asymmetric unit, and the structure was solved by means ofmolecular replacement and refined to a crystallographic R value of 19.7%(FIG. 26 and FIG. 47, Table S1).

The interaction surface between VRC01 and gp120 encompasses almost 2500Å2, with 1244 Å2 contributed by VRC01 and 1249 Å2 by gp120. On VRC01,both heavy chain (894 Å2) and light chain (351 A2) contribute to thecontact surface (FIG. 48, Table S2), with the central focus of bindingon the heavy chain—second complementarity—determining region (CDR H2).Over half of the interaction surface of VRC01 (644 Å2) involves CDR H2,a mode of binding that is reminiscent of the interaction between gp120and the CD4 receptor; CD4 is a member of the V-domain class of theimmunoglobulin superfamily, and the CDR2-like region of CD4 is a centralfocus of gp120 binding (FIG. 27A and FIG. 49, Table S3). For CD4, theCDR2-like region forms antiparallel, intermolecular hydrogen bonds withresidues 365gp120 to 368gp120 of the CD4-binding loop of gp120 (FIG.27B); with VRC01, one hydrogen bond is observed between the carbonyloxygen of Gly54VRC01 and the backbone nitrogen of Asp368gp120. Thishydrogen bond occurs at the loop tip, an extra residue relative to CD4is inserted in the strand, and the rest of the potential hydrogen bondsare of poor geometry or distance (FIG. 27C and FIG. 50, Table S4). Othersimilarities and differences with CD4 are found: Of the two dominant CD4residues (Phe43CD4 and Arg59CD4) involved in interaction with gp120,VRC01 mimics the arginine interaction but not the phenylalanine one(FIG. 27, B and C). Lastly, substantial correlation was observed betweengp120 residues involved in binding VRC01 and CD4 (FIG. 32).

Superposition of the gp120 core in its VRC01— bound form with gp120s inother crystalline lattices and bound by other ligands indicates aCD4-bound conformation [Protein Data Bank (PDB) ID number 3JWD] to bemost closely related in structure, with a Ca-root-mean-square deviationof 1.03 Å (FIG. 51, Table S5). Such superposition of gp120s from CD4-and VRC01-bound conformations brings the N-terminal domain of CD4 andthe heavy chain—variable domain of VRC01 into close alignment (FIG. 27),with 73% of the CD4 N-terminal domain volume overlapping with VRC01.This domain overlap is much higher than observed with the heavy chainsof other CD4-binding-site antibodies, such as b12, b13, or F105 (FIG.52, Table S6). However, when the VRC01 heavy chain is superimposed—onthe basis of conserved framework and cysteine residues—on CD4 in theCD4-gp120 complex, clashes are found between gp120 and the entire topthird of the VRC01 variable light chain (FIG. 27D). In its complex withgp120, VRC01 rotates 43° relative to the CD4-defined orientation andtranslates 6 Å away from the bridging sheet, to a clash-free orientationthat mimics many of the interactions of CD4 with gp120, although withconsiderable variation. Analysis of electrostatics shows that theinteraction surfaces of VRC01 and CD4 are both quite basic, although theresidue types of contacting amino acids are distinct (FIG. 33). Thus,although VRC01 mimics CD4 binding to some extent, considerabledifferences are observed.

When CD4 is placed into an immunoglobulin context by fusing its twoN-terminal domains to a dimeric immunoglobulin constant region, itachieves reasonable neutralization. VRC01, however, neutralizes moreeffectively (FIG. 28A). To understand the structural basis for theexceptional breadth and potency of VRC01, its interactive surface withgp120 was analyzed. VRC01 focuses its binding onto the conformationallyinvariant outer domain, which accounts for 87% of the contact surfacearea of VRC01 (FIG. 53, Table S7). The 13% of the contacts made withflexible inner domain and bridging sheet are noncontiguous, and it wasnoted that these were not critical for binding. In contrast, CD4 makes33% of its contacts with the bridging sheet, and many of theseinteractions are essential. The reduction in inner domain and bridgingsheet interactions by VRC01 is accomplished primarily by a 6Åtranslation relative to CD4, away from these regions; critical contactssuch as made by Phe43CD4 to the nexus of the bridging sheet—outer domainare not found in VRC01, whereas those to the outer domain (such asArg59CD4) are mimicked by VRC01.

To determine the affinity of VRC01 for gp120 in CD4-bound andnon-CD4-bound conformations, surface-plasmon resonance spectroscopy wasused to measure the affinity of VRC01 and other gp120-reactiveantibodies and ligands to two gp120s: a b4-deletion that is restrainedfrom assuming the CD4-bound conformation or a disulfidestabilized gp120core that is largely fixed in the CD4-bound conformation in the absenceof CD4 itself (FIG. 28B and FIG. 34). VRC01 showed high affinity to bothCD4-bound and non-CD4-bound conformations, which is a property shared bythe broadly neutralizing b12 antibody. In contrast, antibodies F105 and17b as well as soluble CD4 showed strong preference for either one, butnot both, of the conformations. To assess the binding of VRC01 in thecontext of the functional viral spike, its ability to neutralizevariants of HIV-1 with gp120 changes that affect the ability to assumethe CD4-bound state was examined. Two of these mutations, His66Ala gp120and Trp69Leugp120, are less sensitive, whereas a third, Ser375Trpgp120,is more sensitive to neutralization by CD4. VRC01 neutralized all threeof these variant HIV-1 viruses with similar potency (FIG. 28C),suggesting that VRC01 recognizes both CD4-bound and non-CD4-boundconformations of the viral spike. This diversity in recognition allowsVRC01 to avoid the conformational masking that hinders mostCD4-binding-site ligands and to potently neutralize HIV-1. Precisetargeting by VRC01. Prior analysis of effective and ineffectiveCD4-binding-site antibodies suggested that precise targeting to thevulnerable site of initial CD4 attachment is required to block viralentry. This site represents the outer domain contact for CD4. Analysisof the VRC01 interaction with gp120 shows that it covers 98% of thissite (FIG. 29, A and B, and FIG. 35), comprising 1089 Å2 on the gp120outer domain, which is about 50% larger than the 730 Å2 surface coveredby CD4. The VRC01 contact surface outside the target site is largelylimited to the conformationally invariant outer domain and avoidsregions of conformational flexibility. This concordance of binding ismuch greater than for ineffective CD4-binding-site antibodies as well asfor those that are partially effective, such as antibody b12 (FIG. 35).The outer domain-contact site for CD4 is shielded by glycan. Contacts bythe VRC01 light chain (Tyr28VRC01 and Ser30VRC01) are made with theprotein-proximal N-acetylglucosamine from the N-linked glycan at residue276gp120. Thus instead of being occluded by glycan, VRC01 makes use of aglycan for binding. Other potential glycan interactions may occur withdifferent strains of HIV-1 because the VRC01 recognition surface on thegp120—outer domain extends further than that of the functionallyconstrained CD4 interaction surface, especially into the loop D and theoften-glycosylated V5 region (FIG. 36).

In addition to conformational masking and glycan shielding, HIV-1resists neutralization by antigenic variation. To understand the basisof this natural resistance to VRC01, 17 resistant isolates were analyzedall by threading their sequences onto the gp120 structure (FIG. 36).Variation was observed in the V5 region in resistant isolates, and thisvariation along with alterations in gp120 loop D appeared to be thesource of most natural resistance to VRC01 (FIG. 29C and FIGS. 36 and37).

Because substantial variation exists in V5, structural differences inthis region might be expected to result in greater than 10% resistance.The lower observed frequency of resistance suggests that VRC01 employs arecognition mechanism that allows for binding despite V5 variation.Examination of VRC01 interaction with V5 shows that VRC01 recognition ofV5 is considerably different from that of CD4 (FIG. 38), with Arg61VRC01in the CDR H2 penetrating into the cavity formed by the V5 and b24strands of gp120 (FIG. 39). The V5 loop fits into the gap between heavyand light chains; thus, by contacting only the more conserved residuesat the loop base, VRC01 can tolerate variation in the tip of the V5 loop(FIG. 29D).

Examination of the structure of VRC01 was conducted for special featuresthat might be required for its function. A number of unusual featureswere apparent, including a high degree of affinity maturation, an extradisulfide bond, a site for N-linked glycosylation, a two-amino-aciddeletion in the light chain, and an extensively matured bindinginterface between VRC01 and gp120 (FIG. 30 and FIG. 40). The frequencywith which these features were found in HIV-1 Env-reactive antibodies orin human antibody-antigen complexes was assessed (FIG. 41, FIG. 54,Table S8 and FIG. 55, Table S9) and measured the effect of genomicreversion of these features on affinity for gp120 and neutralization ofvirus (FIG. 30, A to D, and FIGS. 56 and 57, Table S10).

Higher levels of affinity maturation have been reported forHIV-1—reactive antibodies in general and markedly higher levels forbroadly neutralizing ones. These maturation levels could be a by-productof the persistent nature of HIV-1 infection and may not represent afunctional requirement. Removal of the N-linked glycosylation or theextra disulfide bond, which connects CDR H1 and H3 regions of the heavychain, had little effect on binding or neutralization (FIG. 30, A and B,and FIGS. 56 and 57, Table S10). Insertion of two amino acids to revertthe light chain deletion had moderate effects, which were larger for anAla-Ala insertion (50-fold decrease in binding affinity) versus aSer-Tyr insertion (fivefold decrease in affinity), which mimics thegenomic sequence (FIG. 30C and FIGS. 56 and 57, Table S10). Lastly,reversion of the interface was examined with either single-, four-,seven- or 12-mutant reversions. For the single-mutant reversions of theinterface to the genomic antibody sequence, all 12 mutations had minoreffects [most with a less than two-fold effect on the dissociationconstant (Kd), with the largest effect for a Gly54Ser change having a Kdof 20.2 nM] (FIGS. 56 and 57, Table S10). Larger effects were observedwith multiple (four, seven, or 12) changes (FIG. 30D and FIGS. 56 and57, Table S10). Thus, although VRC01 has a number of unusual features,no single alteration to genomic sequence substantially altered bindingor neutralization.

The probability for elicitation of a particular antibody is a functionof each of the three major steps in B cell maturation: (i) recombinationto produce nascent antibody heavy and light chains from genomic VH-D-Jand Vk/l-J precursors, (ii) deletion of auto-reactive antibodies, and(iii) maturation through hypermutation of the variable domains toenhance antigen affinity. For the recombination step, a lack ofsubstantial CDR L3 and H3 contribution to the VRC01-gp120 interface(FIG. 48, Table S2) indicates that specific Vk/l-J or VH(D)Jrecombination is not required (FIG. 42). The majority of recognitionoccurs with elements encoded in single genomic elements or cassettes,suggesting that specific joining events between them are not required.Within the VH cassette, a number of residues associated with theIGHV1-02*02 precursor of VRC01 interact with gp120; many of these areconserved in related genomic VHs, some of which are of similar geneticdistance from VRC01 (FIGS. 43 and 44). These results suggest thatappropriate genomic precursors for VRC01 are likely to occur at areasonable frequency in the human antibody repertoire.

Recombination produces nascent B cell—presented antibodies that havereactivities against both self and nonself antigens. Those withautoreactivity are removed through clonal deletion. With many of thebroadly neutralizing antibodies to HIV-1, such as 2G12 (glycan reactive)(33, 34), 2F5, and 4E10 (membrane reactive), this appears to be a majorbarrier to elicitation. Although this remains to be characterized forgenomic revertants and maturation intermediates, no autoreactivity hasso far been observed with VRC01.

The third step influencing the elicitation of VRC01-like antibodies isaffinity maturation, which is a process that involves the hypermutationof variable domains combined with affinity-based selection that occursduring B cell maturation in germinal centers. In the case of VRC01, 41residue alterations were observed from the genomic VH gene and 25alterations from the Vk gene (including a deletion of two residues)(FIG. 45). To investigate the effect of affinity maturation on HIV-1gp120 recognition, the VH and Vk regions of VRC01 were reverted, eitherindividually or together, to the sequences of their genomic precursors.The affinity and neutralization of these reverted antibodies was tested(FIG. 31A) and this data was combined with the genomic reversion dataobtained while querying the unusual molecular features of VRC01 (FIG.31B). No antibodies containing VH and Vk regions, which were fullyreverted to their genomic precursors, bound gp120 or neutralized virus.Binding affinity and neutralization showed significant correlations withthe number of affinity matured residues (P<0.0001). Binding tostabilized gp120 did not correlate well with other types of gp120 or toneutralization (FIG. 58, Table S11), which is related in part to greaterretention of binding to VRC01 variants with genomically reverted Vkregions. Extrapolation of the correlation to the putative genomic V genesequences predicted binding affinities of 0.7 T 0.4 mM Kd for gp120stabilized in the CD4-bound conformation and substantially weakeraffinities for nonstabilized gp120s (FIG. 31B and FIG. 46).

No single affinity maturation alteration appeared to affect affinity bymore than ten-fold, suggesting that affinity maturation occurs inmultiple small steps, which collectively enable tight binding to HIV-1gp120. When the effects of VRC01 affinity maturation reversions aremapped to the structure of the VRC01-gp120 complex, they are broadlydistributed throughout the VRC01 variable domains rather than focused onthe VRC01-gp120 interface. Noncontact residues therefore appear toinfluence the interface with gp120 through indirect protein-foldingeffects. Thus, for VRC01 the process of affinity maturation entailsincremental changes of the nascent genomic precursors to obtainhigh-affinity interaction with the HIV-1 Env surface. Receptor mimicryand affinity maturation. The possibility that antibodies use conservedsites of receptor recognition to neutralize viruses effectively has beenpursued for several decades. The recessed canyon on rhinovirus thatrecognizes the unpaired terminal immunoglobulin domains of intercellularadhesion molecule-1 highlights the role that a narrow canyon entrancemay play in such occlusion of bivalent antibody combining regions,although framework recognition can in some instances permit entry.Partial solutions such as those presented by antibody b12(neutralization of ˜40% of circulating isolates) or by antibody HJ16(neutralization of ˜30% of circulating isolates), a recently identifiedCD4-binding-site antibody, may allow recognition of some HIV-1 isolates.

Materials and Methods

Expression and Purification of HIV-1 gp120 Proteins.

The codon-optimized plasmid pVRC8400-HIV-1 Clade A/E 93TH057 ΔV123contains gp120 residues 44-492 with specific deletions at the V1/V2 andV3 regions. The expression construct was made by inserting mouseinterleukin-2 (IL-2) leader sequence (MYSMQLASCVTLTLVLLVN, SEQ ID NO:36) followed by the modified gp120 sequence between the 5′ XbaI and 3′BamHI sites. In the modified gp120 sequence, N-term residues 31-43 ofwild type Clade A/E 93TH057 gp120 were trimmed, amino acids sequences¹²⁴PLCVTLHCTTAKLTNVTNITNVPNIGNITDEVRNCSFNMTTEIRDKKQKVHALFYKLDIVQIEDKNDSSKYRLINCNT¹⁹⁸ (SEQ ID NO: 37) of the V1/V2 loop and³⁰²NMRTSMRIGPGQVFYRTGSIT³²³ (SEQ ID NO: 38) of the V3 loop were replacedwith GG and GGSGSG (SEQ ID NO: 39) linkers, respectively. YU2 Δβ4 coregp120 was constructed with YU2 gp120 sequence by replacing the 9residues of β3-β5 loop with a Gly-Gly linker This deletion weakens thebinding of CD4 and prohibits formation of the antibody 17b epitope. The93TH057, YU24β4 core gp120 and HXBc2 Ds12F123 core gp120 were expressedand purified. Briefly 1 L of HEK 293 GnTi— or 293 FreeStyle cells weretransiently transfected with the mixture of 500 μg of gp120 DNA plasmidand 1 ml of 293fectin (Invitrogen). The transfected cells were incubatedin FreeStyle 293 expression medium (Invitrogen) supplemented with 3%Cell Boost (HyClone) and 2 mM Butyrate (SIGMA) for suspension culture at8% CO2, 37.0° C. and 125 rpm for five days after transfection. Thesupernatants for 93TH057 and HXBc2 Ds12F123 5 core gp120 were harvestedand proteins purified with a protein A-immobilized 17b antibody affinitycolumn. The YU24β4 core gp120 was purified with a protein A-immobilizedF105 antibody affinity column. The gp120 proteins were eluted with IgGelution buffer (Pierce) and immediately adjusted to pH 7.5.

Production of VRC01 IgG and Antigen-Binding Fragment.

The VRC01 IgG was expressed and purified. Briefly, heavy and light chainplasmids were transfected into 293F cells using 293Fectin (Invitrogen).The supernatant was harvested 5 days after transfection, filteredthrough 0.45 μm filter, and followed by purification using immobilizedprotein A or protein G columns. To produce antigen-binding fragments(Fab), VRC01 IgG was incubated at 37° C. with protease Lys-C (Roche) ata ratio IgG:LysC=4000:1 (w/w) in 10 mM EDTA, 100 mM Tris/Cl—, pH 8.5 forabout 12 hours. Uncleaved IgG and the constant fragment (Fc) wereremoved by passing the digestion mixture through a Protein A affinitycolumn; the flowthrough containing VRC01 Fab was concentrated and loadedonto size-exclusion column (Superdex S200) for further purification.

Deglycosylation, Complex Formation and Crystallization of thegp120:VRC01 Complexes.

Deglycosylation of HIV-1 gp120 was performed in a reaction solutioncontaining 1-5 mg/ml gp120, 350 mM NaCl, 100 mM Na Acetate, pH 5.9,1×EDTA-free protease inhibitor (Roche) and endoglycosidase H (30units/μg of gp120). After mixing all components and adjusting pH to 5.9,the solution was incubated at 37° C. and the deglycosylation process wasmonitored by SDS-PAGE until completion. gp120:VRC01 complexes were made.Briefly, VRC01 Fab in 20% molar excess was combined with deglycosylatedgp120 and the gp120:VRC01 mixture was passed through a concanavalin Acolumn to remove gp120 with uncleaved N-linked glycans. The complex wasthen purified by size exclusion chromatography (Hiload 26/60 SuperdexS200 prep grade, GE Healthcare) and concentrated to ˜10 mg/ml in 0.35 MNaCl, 2.5 mM Tris pH 7.0, 0.02% NaN3 for crystallization screeningstudies. To achieve better chances of crystallization hits, two gp120variants, clade B HxBc2 core Ds12F123 and clade A/E 93TH057, were usedto make complexes with the VRC01 Fab. Commercially available screens,Hampton Crystal Screen (Hampton Research), Precipitant Synergy Screen(Emerald BioSystems), and Wizard Screen (Emerald BioSystems), were usedfor initial crystallization trials of the gp120:VRC01 complexes.Vapor-diffusion sitting drops were set up robotically by mixing 0.1 μlof protein with an equal volume of precipitant solutions (Honeybee,DigiLab). Droplets were allowed to equilibrate at 20° C. and imaged atscheduled times with RockImager (Formulatrix.). Multiple crystal hitswere obtained from both HXBc2:VRC01 and 93TH057:VRC01 complexes. Thosehits were optimized manually using the hanging drop vapor-diffusionmethod. Crystals of the HXBc2:VRC01 complex were obtained in 1.0 MNaCltrate, 100 mM NaCacodylate, pH 6.5. For the 93TH057:VRC01 complex,the best condition to obtain diffraction-quality crystals was 10% PEG8000, 100 mM Tris/Cl—, pH 8.5 with 3% glucose as additive.

X-Ray Data Collection, Structure Determination and Refinement for thegp120:VRC01 Complex.

The diffraction of gp120:VRC01 crystals were tested under cryogenicconditions. To search for the best cryo-protectant, protecting effectsof six commonly used cryoprotectants, 30% glycerol, 30% ethylene glycol,15% 2R,3R-butanediol, 40% trihalose, 40% sucrose and 40% glucose, wereassessed. Crystals were transferred into solutions which were composedof crystallization reservoir solution with 50% higher concentration ofprecipitant(s) and each individual cryo-protectant or mixture ofcryo-protectants, immediately flash frozen in liquid nitrogen with acryo-loop (Hampton Research) and mounted under cryo condition)(100K° fordata collection. X-ray data were collected at beam-line ID-22 (SER-CAT)at the Advanced Photon Source, Argonne National Laboratory, with 0.82656Å radiation, processed and reduced with HKL2000). None of theHXBc2:VRC01 crystals diffracted beyond 4 Å resolution and they were notused for data collection. A 2.9 Å data set for the 93TH057:VRC01crystals was collected using a cryoprotectant solutions containing 15%PEG8000, 100 mM Tris/Cl—, pH 8.5 and 20% glucose and 7.5%2R,3R-butanediol as cryoprotectants. I/σ ratio was 1.2 at the 2.9 Åshellwith 68% completeness. The crystal structure of the 93TH057:VRC01complex was solved by molecular replacement with Phaser in the CCP4Program Suite. This crystal belonged to a space group P21 with celldimensions a=108.6, b=98.3, c=205.3, β=99.7 and contained four moleculesper asymmetric unit. The structure of 93TH057 gp120 with β20/β21 regiontrimmed (PDB #3M4M) was used as an initial model to place the gp120 inthe complex. Phaser was able to give a solution with three gp120sinitially (RFZ=4.3 TFZ=4.7 PAK=0 LLG=69 8 RFZ=3.5 TFZ=11.8 PAK=0 LLG=234RFZ=3.7 TFZ=17.5 PAK=0 LLG=485 LLG=1280). With those three gp120s fixed,the CDR-loop-trimmed variable domain (Fv) of antibody b13 (PDB ID 31DX)was used to locate the Fv portion of VRC01 in the complex (RFZ=3.7TFZ=11.7 PAK=0 LLG=539 RFZ=3.9 TFZ=5.0 PAK=0 LLG=389 RFZ=3.9 TFZ=4.7PAK=0 LLG=136 LLG=428). Visual inspection of the generated gp120:Fvsolutions identified one of gp120s complexed with a symmetry-operatedFv. This new gp120:Fv complex was used as model to perform a new roundof molecular replacement, one complex at a time, until all four gp120:Fvcomplexes were found (Round 1: RFZ=6.6 TFZ=8.9 PAK=0 LLG=72 LLG=243,Round 2: RFZ=5.9 TFZ=16.0 PAK=0 LLG=78 LLG=623, Round 3: RFZ=8.4TFZ=25.3 PAK=0 LLG=739 LLG=1866, Round 4: RFZ=5.8 TFZ=22.8 PAK=0 LLG=990LLG=2608). The constant domain of Fab b13 was then used to place one ofthe VRC01 constant domains with all four previous solutions fixed(RFZ=4.7 TFZ=21.5 PAK=1 LLG=1357 LLG=3313). The newly found constantdomain and the gp120:Fv formed a complete 120:VRC01 complex and theother three molecules were generated by superposing this complex withother three gp120s in the asymmetric unit. Further refinement wascarried out with PHENIX. Starting with torsion-angle simulated annealingwith slow cooling, iterative manual model building was carried out onXtalview and COOT with maps generated from combinations of standardpositional, individual B-factor, TLS refinement algorithms andnon-crystallographic symmetry (NCS) restraints. Ordered solvents wereadded during each macro cycle. Throughout the refinement processes, across validation (Rfree) test set consisting of 5% of the data was used.Structure validations were performed periodically during the modelbuilding/refinement process with MolProbity and pdb-care. Even thoughthe reported data at the highest shell of 2.9 Å only has I/σ ratio of1.2, reflections up to 2.7 Å resolution (I/σ>1.0,˜30% completeness) wereincluded and used during the refinement. X-ray crystallographic data andrefinement statistics are summarized in FIG. 47, Table 51.

Surface Plasmon Resonance (SPR).

The binding kinetics of HIV-1 gp120 with different ligands wereperformed on Biacore 3000 or Biacore T-100 (GE Healthcare) at 20° C.with buffer HBS-EP+(10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and0.05% surfactant P-20). To assess VRC01 recognition of gp120 in theCD4-bound and non-CD4-bound conformation, gp120 molecules (YU24(4 coreand HXBc2 Ds12F123 core) were immobilized onto a CM5 chip to 250-500response units (RUs) with standard amine coupling; Fab antibodies, CD4or CD4-IgαTP at 2-fold increasing concentrations were injected over thegp120 channels at a flow rate of 30 μl/min for 3 minutes and allowed todissociate for another 5-10 minutes before regeneration with two 25 μlinjections of 4.5 M MgC12 at a flow rate of 50 μl/ml. To test theeffects of mutations on VRC01, IgG variants were captured with a mouseanti-human IgG Fc antibody supplied in the “human antibody capture kit”(GE Healthcare) to a surface density about 300 RUs. gp120 series with2-fold increasing concentrations were passed over the captured IgG flowchannels for 3 minutes and allowed to dissociate for another 5-10minutes at a flow rate of 30 μl/min. The sensor chip was regeneratedafter each experiment using two 25 μl injections of 4.5 M MgC12 at aflow rate of 50 μl/ml. Sensorgrams were corrected with appropriate blankreferences and fit globally with Biacore Evaluation software using a 1:1Langmuir model of binding. Sensorgrams of IgG b12 binding to gp120 couldnot be fitted with 1:1 Langmuir model and were analyzed with a 10two-state binding model; such treatment should not affect the primaryon-rates nor overall KDs reported here.

Mutagenesis and creation of VRC01 genomic revertants. Single-4-, 7-,12-revertant mutations to germline residues of VRC01 as well as theheavy chain C32SC98A mutations, light chain insertions with Ala-Ala orSer-Tyr after position 30 were listed in FIG. 59, Table S12 and themutagenesis were carried out using Quikchange kit (Stratagene) accordingto manufacturer's protocol. V-gene revertants for VRC01 were constructedas follows: For heavy chain Vgene revertant (gH), VRC01 heavy chainV-gene region was reverted to its germline precursor IGHV 1-02*02. Forlight chain V-gene revertant (gL), VRC01 light chain Vgene region wasreverted to its germline precursor IGKV 3-11*01. The modified heavy andlight chain genes were synthesized by GeneArt (Regensburg, Germany), andcloned into a mammalian CMV/R vector for expression. All the VRC01variants were expressed with the same protocol as wild type VRC01 IgG.

Neutralization Assays.

Neutralization Assays of Viruses by VRC01 and its Variants:

HIV-1 Env-pseudoviruses were prepared by transfecting 293T cells (6×10⁶cells in 50 ml growth medium in a T-175 culture flask) with 10 μg ofrev/env expression plasmid and 30 μg of an env-deficient HIV-1 backbonevector (pSG3ΔEnvelope), using Fugene 6 transfection reagents(Invitrogen). Pseudovirus-containing culture supernatants were harvestedtwo days after transfection, filtered (0.45 μm), and stored at −80° C.or in the vapor 11 phase of liquid nitrogen. Neutralization was measuredusing HIV-1 Env-pseudoviruses to infect TZM-bl cells as describedpreviously. Briefly, 40 μl of virus was incubated for 30 min at 37° C.with 10 μl of serial diluted test antibody in duplicate wells of a96-well flat bottom culture plate. To keep assay conditions constant,sham media was used in place of antibody in specified control wells. Thevirus input was set at a multiplicity of infection of approximately0.01, which generally results in 100,000 to 400,000 relative light units(RLU) in a luciferase assay (Bright Glo, Promega, Madison, Wis.). Theantibody concentrations were defined at the point of incubation withvirus supernatant. Neutralization curves were fit by nonlinearregression using a 5-parameter hill slope equation. The 50% inhibitoryconcentrations (IC50) were reported as the antibody concentrationsrequired to inhibit infection by 50%.

Neutralization Assay of Viruses with Altered Sampling of the CD4-BoundState by VRC01 and CD4.

Recombinant HIV-1 expressing the firefly luciferase gene was produced bycalcium phosphate transfection of 293T cells with the molecular clonepNL4.3 (Env-) Luc and the pSVIIIenv plasmid expressing the wild-type ormutant HIV-1YU2 envelope glycoproteins at a weight ratio of 2:1. Twodays after transfection, the cell supernatants were harvested. Thereverse transcriptase activities of all virus preparations weremeasured. Each virus preparation was frozen and stored in aliquots at−80° C. until use. Luciferase-expressing viruses bearing eitherwild-type or mutant envelope glycoproteins were incubated for 1 hour at37° C. with serial dilutions of sCD4 or VRC01 IgG in a total 12 volumeof 200 μl. The recombinant viruses were then incubated withCf2Th-CD4/CCR5 cells; luciferase activity in the cells was measured twodays later.

ELISA Assay.

Clade A/E 93TH057 and clade B HXBc2 core Ds12F123 gp120 in PBS (pH 7.4)at 2 μg/ml were used to coat plates for 2 hours at room temperature(RT). The plates were washed five times with 0.05% Tween 20 in PBS(PBS-T), blocked with 300 μl per well of block buffer (5% skim milk and2% bovine albumin in PBS-T) for 1 hour at RT. 100 μl of each monoclonalantibodies 5-fold serially diluted in block buffer were added andincubated for 1 hour at RT. Horseradish peroxidase (HRP)-conjugated goatanti-human IgG (H+L) antibody (Jackson ImmunoResearch Laboratories Inc.,West Grove, Pa.) at 1:5,000 was added for 1 hour at RT. The plates werewashed five times with PBS-T and then developed using3,3′,5,5′-tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories)at RT for 10 min. The reaction was stopped by the addition of 100 μl NH2SO4 to each well. The readout was measured at a wavelength of 450 nm.All samples were performed in triplicate.

Analysis of the Commonality of VRC01 Features.

Structural Dataset for Analysis of Antibody Affinity Maturation:

Initially, the IMGT/3Dstructure-DB was searched for structures of humanantibody-protein and antibody-peptide complexes of at most 3.5 Åresolution. This resulted in a set of 54 antibody-protein and 66antibody-peptide structures (the 2wuc complex was found in both theprotein and peptide databases). To complement the structure query, theRSCB PDB was searched for “human fab complex” and structure resolutionof at most 3.5 Å, resulting in a set of 290 structure hits; 211 of thesehad not been identified as part of the IMGT/3Dstructure-DB search. Allunique pdbs from the IMGT/3Dstructure-DB database and the PDB searchwere manually inspected. Additionally, two structures (2b4c and 3hil,see FIG. 58, Table S11) not found in either search were also inspected.To be included in the affinity maturation analysis, pdbs had to possessthe following properties: antibody-protein or antibody-peptide complex,IgG antibody of human origin, natural affinity maturation, and antigennot artificially modified for improved binding. In the cases wheremultiple complexes of the same antibody were identified, only one suchcomplex was used for the analysis. As a result, only 26 of thecomplexes, shown in FIGS. 56 and 57, Table S10, were retained for theantibody affinity maturation analysis. The list of discarded pdbs, alongwith the specific reasons for discarding, is shown in FIG. 58, TableS11. For each of the selected 26 antibody complexes, the number ofantibody contact residues that were mutated from germline was computedfor the antibody V-segments. Contact residues were identified usingPISA. Antibody germline genes were identified with IgBLAST(ncbi.nlm.nih.gov/igblast) using antibody protein sequences for thesearch; insertions/deletions were not counted toward the number ofmutations from germline. A summary of the number of contact residues,V-segment mutated residues, and mutated contact residues for the 26antibodies are shown in FIGS. 56 and 57, Table S10.

Analysis of Cys Residues, Residue Deletions, and Glycan Additions:

A dataset of human HIV-1 antibody heavy and light chain sequences wasobtained. The final curated version of that dataset that excludednon-specific gp140 14 binders as well as sequences withnon-fully-resolved variable regions, included 147 heavy and 147 lightchain sequences. Sequence alignment to germline was performed usingIMGT/V-QUEST. The number of glycans was computed for the V-D-J heavy andV-J light regions. The number of Cys residues was computed for the V-D-Jheavy regions only. The number of residue deletions was computed for theV-segments as compared to the corresponding germline; a deletion ofmultiple consecutive amino acids was counted as a single deletion.

Numbering of Amino Acid Residues in Antibody

the Kabat nomenclature for amino acid sequences in antibodies wasfollowed.

Protein Structure Analysis and Graphical Representations

GRASP and APBS were used in calculations of molecular surfaces, volumes,and electrostatic potentials. PISA was used to perform protein-proteininterfaces analysis. CCP4 was used for structural alignments. Allgraphical representation with protein crystal structures were made withPymol.

Example 3 2F5 Antibody with Enhanced Neutralization Capacity

The membrane-proximal external region (MPER) of the HIV-1 gp41transmembrane glycoprotein is the target of three broadly neutralizinganti-HIV-1 antibodies, 2F5, Z13e, and 4E10, and is thus a potential siteof HIV-1 vulnerability to the humoral immune response. The MPERencompasses ˜25 residues at the carboxyl-terminal end of the predictedgp41 ectodomain, just before the transmembrane region, and is rich inaromatic residues, typical of bilayer-interfacial regions of membraneproteins. Mutation of selected MPER tryptophans abrogates gp41-mediatedfusion of the viral and target cell membranes, indicating that thisregion is crucial for HIV-1 infectivity. Structural studies of unboundforms of the gp41MPER both in solution and in lipid contexts havedemonstrated that it adopts a number of conformations, many of which are_-helical, and electron-paramagnetic resonance measurements haveindicated lipid bilayer immersion depths for MPER residues that rangefrom acyl to phospholipid headgroup regions. The binding of neutralizingantibodies, such as 2F5, to the MPER must therefore account for themembrane milieu in which the epitope is found.

The 2F5 antibody has been shown to exhibit ˜100-fold enhanced binding toits epitope on uncleaved gp140s when presented in the context of lipidproteoliposomes, and other studies have shown that 2F5 can contactphospholipids directly in the absence of gp41. The latter finding hasled to the suggestion that 2F5 might be autoreactive, although passivetransfusion of 2F5 does not appear to have deleterious effects and 2F5failed to react in some clinically based assays for autoreactive lipidantibodies. The crystal structures of the 2F5 antibody in complex withits gp41MPER epitope revealed that, despite the 22-residue length of the2F5 heavy chain third complementarity-determining region (CDR H3) loop,contacts with the gp41MPER peptide are made predominantly at the loopbase. In some crystal structures, the tip of the loop protrudes awayfrom gp41, while in others, it is disordered. A unique feature of thetip of the CDR H3 loop is that it contains a patch of hydrophobicresidues, including residues L100A, F100B, V100D, and HOOF (Kabatnumbering), which, with the exception of HOOF, do not contact gp41 (FIG.62). While a prior study revealed the importance of residue F100B of theCDR H3 loop in 2F5-neutralizing activity, nonconservative residuesubstitutions at this position also appeared to diminish 2F5 binding tothe immobilized MPER peptide and gp41 in enzyme-linked immunosorbentassay (ELISA) formats. Conversely, a more recent study has shown thatalanine mutations in the 2F5 CDR H3 loop can affect neutralizationwithout affecting gp41 binding

As disclosed herein the role of the chemical nature of residues at thetip of the 2F5 CDR H3 loop in neutralization of HIV-1 is examined.Mutations were introduced into the 2F5 CDR H3 loop that altered itshydrophobicity, and the resulting 2F5 mutants were tested both forbinding to a gp41 epitope peptide and for neutralization of HIV-1. Theresults showed that the tip of the 2F5 CDR H3 loop, and specifically itshydrophobic nature, is required for 2F5-mediated neutralization of HIV-1by means that appear to be independent both of gp41 affinity and ofisolatespecific sensitivity to neutralization by 2F5.

Materials and Methods

Antibodies.

The heavy and light chains of the 2F5 antibody were codon optimized formammalian expression, synthesized, and transferred separately into thepVRC8400 (CMV/R) mammalian expression vector. Mutations within the 2F5CDR H3 loop were analyzed for structural compatibility with neighboringresidues and were then introduced into the 2F5 heavy-chain plasmid usingstandard site-directed mutagenesis techniques, implemented by ACGT,Inc., Chicago, Ill. The wild-type and mutant 2F5 heavy- and light-chainplasmids were transiently transfected into 293 Freestyle cells using293fectin (Invitrogen), and supernatants containing secreted IgGs wereharvested 72 to 96 h posttransfection. The IgGs were purified by flowingthe supernatants over a protein A-agarose column (Pierce), followed byelution with IgG elution buffer (Pierce).

Peptides.

A wild-type gp41MPER peptide corresponding to residues 657 to 669 ofgp41 (HxB2 numbering) linked to a C-terminal C9 tag was used and wascomprised of the sequence EQELLELDKWASLGGTETSQVAPA (SEQ ID NO:40)(American Peptide).

Surface Plasmon Resonance.

Biacore 3000 (GE Healthcare) was used in tests. 2F5 wild-type and mutantIgGs were coupled directly to Biacore CMS chips at final densities of˜4,000 to 5,000 response units (RU). The gp41MPER peptide was used asthe analyte and flowed over at 2-fold serial dilutions ranging from 500to 0.49 nM at a flow rate of 30 μl/min for 3 min, followed by injectionof standard Biacore HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mMEDTA, 0.01% P-20) for 3 min. Binding profiles were analyzed using eitherBiaevaluation software (GE Healthcare) or Scrubber version 2 (Biologic).

Virus Neutralization Assays.

A single-cycle infectivity assay using Envpseudotyped virus and Tzm-bltarget cells (NIH AIDS Research and Reference Reagent Program) was usedto assess the neutralization capacities of the 2F5 variants. Env fromthe HIV-1 strains MN, HxB2, JR-FL, SC422661.8, RHPA4259.7, and TRO.11were used, as were Env from the HIV-2 strain 7312a and the HIV-2-HIV-1chimera 7312a-C1. Murine leukemia virus (MuLV) Env was used as anegative control.

Hydrophobicity Analysis.

Analysis of the free energies of partitioning the 2F5 CDR H3 loop tipinto octanol or a lipid bilayer interface was performed using MPex 3.1software (blanco.biomol.uci.edu/mpex). The analysis was performed onresidues 100A to 100F of the 2F5 CDR H3 loop using the Totalizerfunction, with no end groups added and the ΔCONH value set at 0.

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism version 5.0(GraphPad Software Inc.) and Origin 7 (Originlab Corporation).

Results

Predictive measures of hydrophobicity, as determined by scales of freeenergy for partitioning whole residues from water to a lipid bilayerinterface, ΔG_(wif), or to octanol, ΔG_(oct), were used to designmutations that would either disrupt or augment the predictedpartitioning of the 2F5 CDR H3 tip (residues L100A to I100F) (FIG. 62).The amino acid serine, which is found midway on the ΔG_(wif) andΔG_(oct) hydrophobicity scales, was chosen as a single or doublesubstitution in order to achieve a gradual decrease in hydrophobicity(FIG. 62C and FIG. 63, Table 1). Conversely, tryptophan, which is theresidue most favored to partition into the bilayer interface or octanol,was used to augment the hydrophobicity of the loop (FIG. 62C and FIG.63, Table 1).

To ascertain the effect of the CDR H3 tip mutations on binding of 2F5 tothe gp41MPER, the affinities of the expressed 2F5 variants for agp41MPER peptide were determined using surface plasmon resonance. The2F5 wild-type and mutant IgGs were coupled directly to the biosensorsurface, and a gp41 MPER peptide comprised of residues 657 to 669 ofgp41 (isolate HxB2 numbering) linked to a C-terminal C9 tag was used asanalyte, at 2-fold serial dilutions ranging from 500 to 0.49 nM. Asshown in FIG. 63, Table 1 (FIG. 72), all of the mutants maintainednanomolar affinity to the gp41 peptide. In some cases, such as formutants of I100F, there was a moderate reduction in affinity, likely dueto minor contacts made by this residue with gp41.

The 2F5 mutants were then tested for neutralization of thelaboratory-adapted strain HxB2, which is highly sensitive to wild-type2F5. A single-cycle infectivity assay using Env-pseudotyped virus andTzm-bl target cells was employed for this purpose. As shown in FIG. 64,top, even single replacements of hydrophobic residues of the CDR H3 loopwith serine were able to reduce the neutralization capacity of 2F5against HxB2 by several orders of magnitude. The most noticeable singlemutation effect was observed for the F100BS mutation, which led to amore than 500-fold increase in the 50% inhibitory concentration (IC50)relative to wild-type 2F5 (FIG. 64, top, and FIG. 63, Table 1). Thesingle mutations L100AS and I100FS likewise led to 100-fold increases inthe IC50s, while the V100DS mutation increased the IC50 by about15-fold. Introduction of double mutations to serine at the same residuelocations, furthermore, led to complete abrogation of 2F5-mediatedneutralization of HxB2, with neutralization profiles indistinguishablefrom those of the negative control mouse 1D4 anti-rhodopsin antibody(FIG. 64, middle, CDR H3 loop can reduce and completely disrupt 2F5neutralization, despite maintaining nanomolar affinity for gp41.

Since these findings could be the result either of a disruption ofspecific protein contacts made by these residues or of a disruption ofnonspecific interactions mediated by their chemical nature, mutation ofthe same CDR H3 residues to tryptophan was undertaken. Substitutions totryptophan served two purposes: first, they augmented the hydrophobicityand predicted favorability of free energies of transfer from water to abilayer interface or to octanol (ΔG_(wif) and ΔGoct), and second, thebulky nature of the tryptophan side chain had the potential to disruptprotein-protein contacts, should they exist. When tested forneutralization of HxB2, the tryptophan mutants were either commensuratewith 2F5 wild-type neutralization, such as L100_(A)W and F100_(B)W, orwere even more potent, such as V100_(D)W and the double mutant L100_(A)WV100_(D)W, both of which showed an ˜10-fold decrease in theneutralization IC50 relative to wild-type 2F5 (FIG. 64, bottom).

To rule out the possibility that these results were specific to the HxB2isolate, the 2F5 mutants were tested for neutralization of a panel oftier 1 and tier 2 HIV-1 isolates, ranging from highly sensitive strains,such as MN, to more resistant ones, such as JR-F1, and also to anHIV-2-HIV-1 2F5 epitope chimera, 7312a-C1. As shown in FIG. 63, Table 1,single mutations that decreased the hydrophobicity of the 2F5 CDR H3loop led to decreases in neutralization potency, and double mutationsthat decreased hydrophobicity completely abrogated neutralization.Likewise, single and double mutations that increased the hydrophobicityof the loop led to increased neutralization potencies (FIG. 63, Table1). Meanwhile, neutralization of the Tro.11 isolate, which has a K665Spoint mutation in the core of the epitope, was virtually undetectablefor all 2F5 mutants, similar to what was observed for virusespseudotyped with MuLV and parental HIV-2 7312a Env, which were used asnegative controls.

To determine if there was a statistical relationship between thehydrophobicity of the 2F5 CDR H3 loop and the neutralization capacity ofthe antibody, neutralization IC50s of the 2F5 mutants were plottedagainst the estimated free energies of transfer, ΔG_(wif), of the mutantCDR H3s for each of the strains tested. As shown in FIG. 65A and FIG.66, Table 2, with the exception of RHPA-4259, statistically significantlinear relationships were observed between neutralization IC50s and CDRH3 ΔG_(wif), with P values ranging from 0.0012 for HxB2 to 0.018 forJR-FL. Linear relationships with even more stringent P values wereobserved between neutralization IC50s and the predicted free energy oftransfer to octanol, ΔG_(oct) (FIG. 73A and FIG. 69, Table S1).Associations between the neutralization IC50s and the affinities of thevarious 2F5 mutants for gp41MPER peptide were also examined. As shown inFIG. 65B and FIG. 66, Table 2, despite the fact that the fits appear tobe largely driven by the KD (dissociation constant) of the HOOFS 2F5mutant for gp41 (which makes minor contacts with the peptide), nostatistically significant relationships were observed between theneutralization IC50s of the 2F5 CDR H3 variants and their affinities forthe gp41MPER peptide.

Though the overall levels of the IC50s varied per strain tested, likelya reflection of the strain sensitivity to 2F5 itself, the mutationsappeared to exhibit similar effects on neutralization regardless of thestrain used. Specifically, no significant differences were observed inthe slopes of the linear fits of the neutralization IC50s versusΔG_(wif) or versus ΔG_(oct), unlike their y intercepts, which diddisplay significant differences (FIG. 65A, and FIG. 66, Table 2). Thesimilarities in the slopes of the regressions across all strainssuggested that the neutralization effects mediated by 2F5 CDR H3 loophydrophobicity were largely independent of strain sensitivity to 2F5.The free energy of antibody neutralization (ΔG^(N)) can be viewed as asum of free energies that contribute to its functional interactions witha specific strain of virus. It can also be viewed as a bindingassociation of an antibody “n” with the virion: ΔG^(N)-RT In K(n), whereK(n) is defined as IC50/ƒ(n) and ƒ(n) is a function that accounts forvariables such as strain sensitivity and assay used. Although anabsolute value for this free energy requires the definition of ƒ(n), ifƒ(n1) can be approximated to equal ƒ(n2) for two variants n1 and n2 ofthe same antibody neutralizing the same strain of virus, a relative freeenergy, ΔΔG^(N), can be obtained. In the case of 2F5, it was verifiedhow the free energy of partitioning the CDR H3 loop from water to alipid bilayer interface, ΔG_(wif), or to octanol, ΔG_(oct), correlatedwith the relative free energy of neutralization (ΔΔG^(N)=ΔG_(2F5wt)^(N)−ΔG_(2F5mut) ^(N)). Linear models were used to fit curves of ΔΔG^(N)versus ΔG_(wif) (FIG. 65C) or ΔG_(oct) (FIG. 73C) for each individualstrain and for all strains together. The shared correlations obtainedwere statistically significant, with P<0.0001, although end points athigh hydrophobicity suggested linear fits might not be ideal (seebelow). Nonetheless, the results confirmed that the effects of ΔG_(wif)and ΔG_(oct) of the 2F5 CDR H3 loop on virus neutralization were largelyindependent of the virus strain, with the normalization relative to 2F5wild type in the ΔΔG^(N) calculation making the fits for all the strainsvirtually superimposable (FIG. 65C and FIG. 73C). The shared slopes fromthese correlations were 1.3 and 1.2 for ΔG_(wif) and ΔG_(oct),respectively. Thus, the calculated change in the free energy ofpartitioning the 2F5 CDR H3 loop translates almost directly into changesin neutralization. The 30% enhancement in neutralization, ΔΔG^(N), overthe calculated partition free energy may reflect the planar positioningof these residues in the CDR H3 structure and/or their positioninginduced by recognition of the gp41 protein component.

Though linear regressions provided a reasonable first approximation ofthe relationship between neutralization IC50s and the free energy ofhydrophobic transfer of the tip of the 2F5 CDR H3 loop, it was observedthat beyond a certain threshold of loop hydrophobicity, the effects onneutralization appeared to level off. To account for this observation, aquadratic term was added to the linear regressions. This yielded betterfits of the data, as judged by an extra sums-of-squares F test (FIG.67A, FIG. 73B and FIG. 70, Table S2). Based on these quadratic models,IC50 minima were interpolated for each of the strains tested (FIG. 71,Table S3). On average, the interpolated minimum IC50s were approximately0.96 log units or 9.2-fold lower than the corresponding experimentalIC50s of wild-type 2F5 (FIG. 71, Table S3). Compared to the interpolatedIC50s corresponding to a ΔG_(wif) of 0, in which no transfer ispredicted to occur, the mean minimum IC50s were approximately 5.1 logunits or 13.000-fold lower than those predicted for a 2F5 variant withno capacity for hydrophobic transfer (FIG. 71, Table S3). A quadraticterm was also added to the fits of the plots of the relative freeenergies of neutralization, ΔΔG^(N), versus the predicted free energiesof transfer of the 2F5 CDR H3 loop to a bilayer interface, ΔG_(wif), oroctanol, ΔG_(oct), (FIG. 67B and FIG. 77D). Shared quadratic fits ofΔΔG^(N) versus ΔG_(wif) and ΔG_(oct) were also performed, and ΔΔG^(N)minima were observed at 2F5 CDR H3 ΔG_(wif) and ΔG_(oct) values of −4.08and −5.69 kcal/mol, with corresponding ΔΔG^(N) values of −1.28 and −1.18kcal/mol, respectively (FIG. 67C and FIG. 77D).

Discussion

The results presented herein suggest that in addition to gp41MPERbinding, interactions mediated by the tip of the 2F5 CDR H3 loop arealso required for 2F5-mediated neutralization of HIV-1. For the elementsof 2F5-mediated neutralization described here, the free energy of2F5-mediated virus neutralization, ΔG^(N), can thus be viewed as the sumof the free energy of 2F5 structure-specific recognition of gp41combined with the free energy of transfer of its CDR H3 loop into ahydrophobic milieu (FIG. 68). While a number of possibilities exist toexplain how the tip of the 2F5 CDR H3 mediates neutralization of HIV-1,the finding that mutations to tryptophan were tolerated at threeseparate locations within the CDR H3 loop, in some cases even augmenting2F5-mediated neutralization, is consistent with the loop mediatingnonspecific hydrophobic interactions. It was surmised that suchinteractions are more likely to occur at a lipid bilayer interface thanwithin a protein-protein interface, though 2F5 has been shown totolerate a great deal of sequence variation at its interface with gp41.The finding that correlations of hydrophobicity of the loop andneutralization capacity are largely independent of HIV-1 isolatesensitivity to 2F5, furthermore, suggests that the contacts mediated bythe 2F5 CDR H3 loop are distinct from gp41 binding, at least in terms ofelements of gp41 not conserved across all strains. The fact that this istrue not only for HIV-1 isolates, but also for divergent simianimmunodeficiency virus (SIV)-HIV-1 and HIV-2-HIV-1 chimeras, providesadditional evidence that 2F5 CDR H3 interactions are not specific forHIV-1 envelope. Because the hydrophobic tip of the CDR H3 representsonly a small portion of the surface of antibody 2F5, it seems likelythat the reduction in direct binding to lipid vesicles represents anaveraging of the alteration in CDR H3 tip hydrophobicity relative to theentire 2F5 antibody. It thus seems reasonable to expect that the fulleffects of the mutations described herein for neutralization can likelybe recapitulated in in vitro lipid binding assays only if the 2F5 CDR H3loop is properly oriented relative to gp41 and the viral membrane, as itis in the virion/neutralization context, or when the gp41MPER ispresented in a proteoliposome context. The difference between anoriented CDR H3 effect (large and significant) and an overall effect ondirect biding to lipid vesicles (weak and less significant) may providean explanation for the lack of 2F5 autoreactivity in in vivo studies; inthe former case, the effect is amplified by the precise orientation ofthe CDR H3 to the viral membrane through binding to the proteincomponent of the MPER epitope, while in the latter case, the effect isminimized by entropic effects and by averaging over the entire surfaceof the 2F5 antibody. Finally, it was noted that a number of proteinshave interfacial binding properties similar to those proposed for 2F5.Soluble phospholipases A2, for instance, show dramatic interfacialactivation of catalytic activity and require attachment to membranes toappropriately position a substrate for catalysis. Such systems may allowadditional insight into the neutralization mechanism of 2F5. The sizesand hydrophobicities of likely membrane attachment surfaces, forexample, are decreased in neurotoxic phospholipases A2, which need toavoid nonspecific membrane interactions during diffusion to the neuronalsynapse. Indeed, quadratic fits of our neutralization versus CDR H3hydrophobicity data resulted in improved correlations and appeared toreveal a threshold beyond which additional hydrophobicity did notenhance neutralization. Overall, these findings have numerousimplications for optimization of 2F5 potency and for recreating 2F5-likeantibodies in vaccine settings. Variants of 2F5 with tryptophansubstitutions are already ˜10-fold more potent than the wild type interms of neutralization, and future designs of 2F5-based vaccineimmunogens may thus need to account not only for structure-specificrecognition of gp41 but also for the hydrophobic interactions mediatedby the tip of the 2F5 CDR H3 loop.

Example 4 HIV-1 Monoclonal Neutralizing Antibodies Specific to gp120 orgp41 for Detecting HIV-1 in a Subject

This example describes the use of HIV-1 monoclonal neutralizingantibodies specific to gp120 or gp41 for the detection of HIV-1 in asubject. This example further describes the use of these antibodies toconfirm the diagnosis of HIV-1 in a subject.

A biological sample, such as a blood sample is obtained from the patientdiagnosed with, or suspected of having an HIV-1 infection. A bloodsample taken from a patient who is not infected is used as a control. AnELISA is performed to detect the presence of HIV-1 in the blood sample.Proteins present in the blood samples (the patient sample and controlsample) are immobilized on a solid support, such as a 96-well plate,according to methods well known in the art (see, for example, Robinsonet al., Lancet 362:1612-1616, 2003, incorporated herein by reference).Following immobilization, HIV-1 monoclonal neutralizing antibodiesspecific to gp120 or gp41 that is directly labeled with a fluorescentmarker is applied to the protein-immobilized plate. The plate is washedin an appropriate buffer, such as PBS, to remove any unbound antibodyand to minimize non-specific binding of antibody. Fluorescence can bedetected using a fluorometric plate reader according to standardmethods. An increase in fluorescence intensity of the patient sample,relative to the control sample, indicates the anti-gp120 or gp41antibody specifically bound proteins from the blood sample, thusdetecting the presence of HIV-1 protein in the sample. Detection ofHIV-1 protein in the patient sample indicates the patient has HIV-1, orconfirms diagnosis of HIV-1 in the subject.

Example 5

HIV-1 Monoclonal Neutralizing Antibodies Specific to gp120 or gp41 forthe Treatment of HIV-1

This example describes a particular method that can be used to treat HIVin a human subject by administration of one or more gp120 or gp 41specific human neutralizing mAbs. Although particular methods, dosages,and modes of administrations are provided, one skilled in the art willappreciate that variations can be made without substantially affectingthe treatment.

Based upon the teaching disclosed herein HIV-1 can be treated byadministering a therapeutically effective amount of one or more of theneutralizing mAbs described herein, thereby reducing or eliminating HIVinfection.

Screening Subjects

In particular examples, the subject is first screened to determine ifthey have HIV. Examples of methods that can be used to screen for HIVinclude a combination of measuring a subject's CD4+ T cell count and thelevel of HIV in serum blood levels. Additional methods using the gp120-and gp41-specific mAbs described herein can also be used to screen forHIV.

In some examples, HIV testing consists of initial screening with anenzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV,such as to HIV-1. Specimens with a nonreactive result from the initialELISA are considered HIV-negative unless new exposure to an infectedpartner or partner of unknown HIV status has occurred. Specimens with areactive ELISA result are retested in duplicate. If the result of eitherduplicate test is reactive, the specimen is reported as repeatedlyreactive and undergoes confirmatory testing with a more specificsupplemental test (e.g., Western blot or an immunofluorescence assay(IFA)). Specimens that are repeatedly reactive by ELISA and positive byIFA or reactive by Western blot are considered HIV-positive andindicative of HIV infection. Specimens that are repeatedlyELISA-reactive occasionally provide an indeterminate Western blotresult, which may be either an incomplete antibody response to HIV in aninfected person, or nonspecific reactions in an uninfected person. IFAcan be used to confirm infection in these ambiguous cases. In someinstances, a second specimen will be collected more than a month laterand retested for subjects with indeterminate Western blot results. Inadditional examples, nucleic acid testing (e.g., viral RNA or proviralDNA amplification method) can also help diagnosis in certain situations.

The detection of HIV in a subject's blood is indicative that the subjecthas HIV and is a candidate for receiving the therapeutic compositionsdisclosed herein. Moreover, detection of a CD4+ T cell count below 350per microliter, such as 200 cells per microliter, is also indicativethat the subject is likely to have HIV.

Pre-screening is not required prior to administration of the therapeuticcompositions disclosed herein

Pre-Treatment of Subjects

In particular examples, the subject is treated prior to administrationof a therapeutic agent that includes one or more antiretroviraltherapies known to those of skill in the art. However, suchpre-treatment is not always required, and can be determined by a skilledclinician.

Administration of Therapeutic Compositions

Following subject selection, a therapeutically effective dose of a gp120or gp41 specific neutralizing mAb described herein is administered tothe subject (such as an adult human or a newborn infant either at riskfor contracting HIV or known to be infected with HIV). Additionalagents, such as anti-viral agents, can also be administered to thesubject simultaneously or prior to or following administration of thedisclosed agents. Administration can be achieved by any method known inthe art, such as oral administration, inhalation, intravenous,intramuscular, intraperitoneal, or subcutaneous.

The amount of the composition administered to prevent, reduce, inhibit,and/or treat HIV or a condition associated with it depends on thesubject being treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. Ideally, atherapeutically effective amount of an agent is the amount sufficient toprevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV)in a subject without causing a substantial cytotoxic effect in thesubject. An effective amount can be readily determined by one skilled inthe art, for example using routine trials establishing dose responsecurves. As such, these compositions may be formulated with an inertdiluent or with an pharmaceutically acceptable carrier.

In one specific example, antibodies are administered at 5 mg per kgevery two weeks or 10 mg per kg every two weeks depending upon theparticular stage of HIV. In an example, the antibodies are administeredcontinuously. In another example, antibodies or antibody fragments areadministered at 50 μg per kg given twice a week for 2 to 3 weeks.

Administration of the therapeutic compositions can be taken long term(for example over a period of months or years).

Assessment

Following the administration of one or more therapies, subjects havingHIV can be monitored for reductions in HIV levels, increases in asubjects CD4+ T cell count, or reductions in one or more clinicalsymptoms associated with HIV. In particular examples, subjects areanalyzed one or more times, starting 7 days following treatment.Subjects can be monitored using any method known in the art. Forexample, biological samples from the subject, including blood, can beobtained and alterations in HIV or CD4+ T cell levels evaluated.

Additional Treatments

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of agents that theypreviously received for the desired amount of time, including theduration of a subject's lifetime. A partial response is a reduction,such as at least a 10%, at least 20%, at least 30%, at least 40%, atleast 50%, or at least 70% in HIV infection, HIV replication orcombination thereof. A partial response may also be an increase in CD4+T cell count such as at least 350 T cells per microliter.

Example 6 Use of Epitope Scaffolds as Immunological Probes

In order to detect and isolate structure specific antibodies againstviral neutralizing determinants, use of heterologous epitope scaffoldscan be used.

Epitope scaffolds are proteins that are surface engrafted with anepitope of choice in a manner that maintains the desired structuralconformation of the epitope. In the case of gp41 epitope scaffolds, thegp41 membrane proximal extracellular region (MPER) target of the broadlyneutralizing 2F5 antibody is engrafted onto the surface of heterologousproteins in a manner that preserves its 2F5-bound conformation. Fivesuch scaffolds have been developed thus far using a graft that isderived from a clade B virus. Epitope scaffolds with MPER grafts derivedfrom other clades, such as clades C and A and any other clades or gp41sequences that should be required, are also included as immunologicalprobes in this disclosure. Point mutations within the grafts which knockout binding to 2F5 are also included for use as negative selectionagents—in that isolated antibodies directed against the core of the MPERepitope should not be able to bind to these point mutants.

When used as probes, the gp41MPER epitope scaffolds are biotinylated aspreviously described (Doria-Rose, et al, Journal of Virology, January2009, p. 188-199, Vol. 83, No. 1). An Avitag sequence for biotinylation(LNDIPLAQKIEWHE, SEQ ID NO: 26) is added at the c-terminal end of thescaffold (see sequences below). After expression in either mammaliancells (e.g., 293 freestyle cells, Invitrogen) or in E. coli cells (e.g.,BL21 cells, Novagen), Biotin ligase Bir A (Avidity, Denver, Colo.) isused to biotinylate the scaffolds at the Avitag sequence.

Sequences of gp41MPER Epitope Scaffold Immunological Probes (graft areunderlined and in bold):

ES1 (1LGYA) ES1 (1LGYA) Clade B (SEQ ID NO: 11): EVLEADKWAILGATKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPANLSIFTVGGPRVGNPTFAYYVESTGIPFARTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQVCGSAIETKDCSNSIVPFTSILDHLSYFDINEGSCLSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHEES1 (1LGYA) Clade B point mutant (SEQ ID NO: 12): EVLEADEWAILGATKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPANLSIFTVGGPRVGNPTFAYYVESTGIPFARTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQVCGSAIETKDCSNSIVPFTSILDHLSYFDINEGSCLSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES1 (1LGYA) Clade C (SEQ ID NO: 13):EVLALDSWKNLG ATKYAGIAATAYCRSVVPGNKWDCVQCQKWVPDGKIITTFTSLLSDTNGYVLRSDKQKTIYLVFRGTNSFRSAITDIVFNFSDYKPVKGAKVHAGFLSSYEQVVNDYFPVVQEQLTAHPTYKVIVTGHSLGGAQALLAGMDLYQREPRLSPANLSIFTVGGPRVGNPTFAYYVESTGIPFARTVHKRDIVPHVPPQSFGFLHPGVESWIKSGTSNVQVCGSAIETKDCSNSIVPFTSILDHLSYFDINEGSCLSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES2 (1KU2A-s)ES2 (1KU2A-s) Clade B (SEQ ID NO: 14): ASDPVRQYLHEIG EVLELDKWAELGAAAKVEEGMEAIKKLSEATGLDQELIREVVRAKILGTAAIQKIPGLKEKPDPKTVEEVDGKLKSLPKELKRYLHIAREGEAARQHLIEANLRLVVSIAKKYTGRGLSFLDLIQEGNQGLIRAVEKFEYKRGFAFSTYATWWIRQAINRAIADQARSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES2 (1KU2A-s) Clade B point mutant (SEQ ID NO: 15):ASDPVRQYLHEIG EVLELDEWAELG AAAKVEEGMEAIKKLSEATGLDQELIREVVRAKILGTAAIQKIPGLKEKPDPKTVEEVDGKLKSLPKELKRYLHIAREGEAARQHLIEANLRLVVSIAKKYTGRGLSFLDLIQEGNQGLIRAVEKFEYKRGFAFSTYATWWIRQAINRAIADQARSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES2 (1KU2A-s) Clade C (SEQ ID NO: 16): ASDPVRQYLHEIGEVLALDSWKNLG AAAKVEEGMEAIKKLSEATGLDQELIREVVRAKILGTAAIQKIPGLKEKPDPKTVEEVDGKLKSLPKELKRYLHIAREGEAARQHLIEANLRLVVSIAKKYTGRGLSFLDLIQEGNQGLIRAVEKFEYKRGFAFSTYATWWIRQAINRAIADQARSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES3 (2MATA) ES3 (2MATA) Clade B (SEQ ID NO: 17):EILELDKWAILG MRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKAGAHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGGGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHDSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHEES3 (2MATA) Clade B point mutant (SEQ ID NO: 18): EILELDEWAILGMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKAGAHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGGGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHDSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES3 (2MATA) Clade C (SEQ ID NO: 19):EILALDSWKNLG MRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKAGAHGDTSKMFIVGKPTIMGERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGGGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHDSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES4 (1IWLA) ES4 (1IWLA) Clade B (SEQ ID NO: 20):DAASDLKSRLDKVSSFGAGFTQKVTDVQEGQGALAVKRPNLFAWHMTQPDESILVSDGKTLWFYNPFVEQATATWLKDATGNTPFMLIARNQSSDWQQYNIKQNGDDFVLTPKASNGNLKQFTINVGRDGTIHQFSAVEQDDQRSSYQLKAQ ENLEVDKWAFLF GPPQGVTVDDQRKSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES4 (1IWLA) Clade B point mutant (SEQ ID NO: 21):DAASDLKSRLDKVSSFGAGFTQKVTDVQEGQGALAVKRPNLFAWHMTQPDESILVSDGKTLWFYNPFVEQATATWLKDATGNTPFMLIARNQSSDWQQYNIKQNGDDFVLTPKASNGNLKQFTINVGRDGTIHQFSAVEQDDQRSSYQLKAQ ENLEVDEWAFLF GPPQGVTVDDQRKSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES4 (1IWLA) Clade C (SEQ ID NO: 22):DAASDLKSRLDKVSSFGAGFTQKVTDVQEGQGALAVKRPNLFAWHMTQPDESILVSDGKTLWFYNPFVEQATATWLKDATGNTPFMLIARNQSSDWQQYNIKQNGDDFVLTPKASNGNLKQFTINVGRDGTIHQFSAVEQDDQRSSYQLKAQ ENLAVDSWKNLF GPPQGVTVDDQRKSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE ES5 (1D3BB) ES5 (1D3BB) Clade B (SEQ ID NO: 23):SKMLQHIDYRMRCIGGAGGIAIGTFKAFGAGMGLILCDCDAFAKIKPKNSKQAEREEKAVG ELLELDKWALL SMTVEGPPPSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHEES5 (1D3BB) Clade B point mutant (SEQ ID NO: 24):SKMLQHIDYRMRCIGGAGGIAIGTFKAFGAGMGLILCDCDAFAKIKPKNSKQAEREEKAVG ELLELDEWALL SMTVEGPPPSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHEES5 (1D3BB) Clade C (SEQ ID NO: 25):SKMLQHIDYRMRCIGGAGGIAIGTFKAFGAGMGLILCDCDAFAKIKPKNSKQAEREEKAVG ELLALDSWKNL SMTVEGPPPSGLVPRGSGSHHHHHHGGLNDIFEAQKIEWHE

Example 7

Identification of VCR1-like Antibodies

To demonstrate that light heavy chain complementation can be used toidentify a VR01, VCR03-like antibody, VRC01/VRC03 chimera were createdand expressed (see FIG. 76). The chimeric antibodies were tested forneutralization of HIV constructs (see FIGS. 77-79). The results of thechimeric neutralization studies demonstrated that complementationpredicts an antibody as VRC01 and VRC03-like antibodies. Thuscomplementation is applied to antibody heavy chain sequences generatedby 454 sequencing.

454 sequencing was carried out on a sample obtained from subject 45, and200,000 heavy chain sequences were sorted through these based onthreading and retention of specific residues associated with VRC01 andVRC03 to identify VRC01 and VRC03 like antibody sequence. 700 sequences,with a variable sequence identity to VRC01 and VRC03 were obtained. Toevaluate whether or not these antibodies can adopt the VRC03 bindingmode, their sequences have been “threaded” onto the crystal structure ofVRC03/gp120 complex and a so-called threading score was calculated foreach of the sequences.

The use of threading technique allow incorporation the structuralinformation into the 454 sequencing analysis and to select the sequencesthat can potentially adopt the desired binding mode, which has beenprecisely characterized by the three structures of antibody/gp120complexes (see Wu et al. Rational Design of Envelope Identifies BroadlyNeutralizing Human Monoclonal Antibodies to HIV-1,” Science 329, 856-861(2010), which is incorporated herein by reference in its entirety andZhou et al., “Structural Basis for Broad and Potent Neutralization ofHIV-1 by Antibody VRC01, Science 329, 811-817 (2010), which isincorporated herein by reference in its entirety). The threading scoresrange from 0.07 to 0.13, which are in the lower range of the valuescalculated from the threading of VRC01 and VRC03 and onto each other(The lower the threading score is, the better the sequence matches thestructure). This result indicates that these antibodies are beVRC01-like.

A sequence alignment of one of these antibody heavy chains (AntibodyHeavy Chain No:57203) is shown in FIG. 80 compared to the amino acidsequences of the heavy chains of VRC01, VRC02, and VRC03 and thegermline sequence IGHV1-02*02.1N Antibody Heavy Chain No:57203 wassynthesize and a chimera between this heavy chain and the light chain ofVRC01 was created. These antibodies were tested for neutralization, whencombined with VRC01 light chain (see FIG. 81A-81H). Thus, as disclosedherein, a class of VRC01-like antibodies (heavy chain variable domainonly) has been identified.

Below is a list of the heavy chains of VRC01 and VRC03-like antibodiesidentified. Shown are assigned number of the heavy chain, the deducedgermline (germ), the percent identity to VRC01 (seqid1), the percentidentity to VRC03 (seqid3), the threading value (thrd1), the divergencefrom the germline sequence (divg), the nucleic acid sequence identifierand the amino acid sequence identifier.

VRC01 and VRC03-like Antibodies Antibody Heavy Chain No:81364germ=IGHV1-2*02 seqid1=54.5 seqid3=92.3 thrd1=0.259 thrd3=0.092divg=0.304 Nucleic acid sequence=SEQ ID NO: 60 Amino acid sequence=SEQID NO: 760; Antibody Heavy Chain No:140214 germ=IGHV1-2*02 seqid1=58.7seqid3=99.2 thrd1=0.305 thrd3=0.031 divg=0.297 Nucleic acid sequence=SEQID NO: 61 Amino acid sequence=SEQ ID NO: 761; Antibody Heavy ChainNo:64244 germ=IGHV1-2*02 seqid1=59.5 seqid3=99.2 thrd1=0.350 thrd3=0.111divg=0.297 Nucleic acid sequence=SEQ ID NO: 62 Amino acid sequence=SEQID NO: 762; Antibody Heavy Chain No:30181 germ=IGHV1-2*02 seqid1=54.5seqid3=88.5 thrd1=0.311 thrd3=0.081 divg=0.297 Nucleic acid sequence=SEQID NO: 63 Amino acid sequence=SEQ ID NO: 763; Antibody Heavy ChainNo:22829 germ=IGHV1-2*02 seqid1=59.5 seqid3=100.0 thrd1=0.310thrd3=0.031 divg=0.294 Nucleic acid sequence=SEQ ID NO: 64 Amino acidsequence=SEQ ID NO: 764; Antibody Heavy Chain No:34065 germ=IGHV1-2*02seqid1=59.5 seqid3=100.0 thrd1=0.310 thrd3=0.031 divg=0.294 Nucleic acidsequence=SEQ ID NO: 65 Amino acid sequence=SEQ ID NO: 765; AntibodyHeavy Chain No:42537 germ=IGHV1-2*02 seqid1=59.5 seqid3=100.0thrd1=0.310 thrd3=0.031 divg=0.294 Nucleic acid sequence=SEQ ID NO: 66Amino acid sequence=SEQ ID NO: 766; Antibody Heavy Chain No:37825germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.167 thrd3=0.140divg=0.209 Nucleic acid sequence=SEQ ID NO: 67 Amino acid sequence=SEQID NO: 767; Antibody Heavy Chain No:12853 germ=IGHV1-2*02 seqid1=47.1seqid3=42.3 thrd1=0.206 thrd3=0.230 divg=0.209 Nucleic acid sequence=SEQID NO: 68 Amino acid sequence=SEQ ID NO: 768; Antibody Heavy ChainNo:100463 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.173thrd3=0.126 divg=0.209 Nucleic acid sequence=SEQ ID NO: 69 Amino acidsequence=SEQ ID NO: 769; Antibody Heavy Chain No:127918 germ=IGHV1-2*02seqid1=43.0 seqid3=43.1 thrd1=0.211 thrd3=0.142 divg=0.209 Nucleic acidsequence=SEQ ID NO: 70 Amino acid sequence=SEQ ID NO: 770; AntibodyHeavy Chain No:25055 germ=IGHV1-2*02 seqid1=57.9 seqid3=51.5 thrd1=0.203thrd3=0.211 divg=0.206 Nucleic acid sequence=SEQ ID NO: 71 Amino acidsequence=SEQ ID NO: 771; Antibody Heavy Chain No:92641 germ=IGHV1-2*02seqid1=48.8 seqid3=45.4 thrd1=0.199 thrd3=0.242 divg=0.199 Nucleic acidsequence=SEQ ID NO: 72 Amino acid sequence=SEQ ID NO: 772; AntibodyHeavy Chain No:24567 germ=IGHV1-2*02 seqid1=48.8 seqid3=43.1 thrd1=0.142thrd3=0.124 divg=0.196 Nucleic acid sequence=SEQ ID NO: 73 Amino acidsequence=SEQ ID NO: 773; Antibody Heavy Chain No:60412 germ=IGHV1-2*02seqid1=47.9 seqid3=41.5 thrd1=0.164 thrd3=0.156 divg=0.196 Nucleic acidsequence=SEQ ID NO: 74 Amino acid sequence=SEQ ID NO: 774; AntibodyHeavy Chain No:41501 germ=IGHV1-2*02 seqid1=48.8 seqid3=43.1 thrd1=0.142thrd3=0.124 divg=0.193 Nucleic acid sequence=SEQ ID NO: 75 Amino acidsequence=SEQ ID NO: 775; Antibody Heavy Chain No:25764 germ=IGHV1-2*02seqid1=49.6 seqid3=42.3 thrd1=0.192 thrd3=0.136 divg=0.193 Nucleic acidsequence=SEQ ID NO: 76 Amino acid sequence=SEQ ID NO: 776; AntibodyHeavy Chain No:25287 germ=IGHV1-2*02 seqid1=47.1 seqid3=44.6 thrd1=0.143thrd3=0.229 divg=0.189 Nucleic acid sequence=SEQ ID NO: 77 Amino acidsequence=SEQ ID NO: 777; Antibody Heavy Chain No:25949 germ=IGHV1-2*02seqid1=48.8 seqid3=44.6 thrd1=0.191 thrd3=0.145 divg=0.186 Nucleic acidsequence=SEQ ID NO: 78 Amino acid sequence=SEQ ID NO: 778; AntibodyHeavy Chain No:26177 germ=IGHV1-2*02 seqid1=47.1 seqid3=43.8 thrd1=0.209thrd3=0.169 divg=0.186 Nucleic acid sequence=SEQ ID NO: 79 Amino acidsequence=SEQ ID NO: 779; Antibody Heavy Chain No:32644 germ=IGHV1-2*02seqid1=47.9 seqid3=45.4 thrd1=0.161 thrd3=0.166 divg=0.182 Nucleic acidsequence=SEQ ID NO: 80 Amino acid sequence=SEQ ID NO: 780; AntibodyHeavy Chain No:62663 germ=IGHV1-2*02 seqid1=47.9 seqid3=45.4 thrd1=0.161thrd3=0.166 divg=0.182 Nucleic acid sequence=SEQ ID NO: 81 Amino acidsequence=SEQ ID NO: 781; Antibody Heavy Chain No:19531 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094 divg=0.182 Nucleic acidsequence=SEQ ID NO: 82 Amino acid sequence=SEQ ID NO: 782; AntibodyHeavy Chain No:18715 germ=IGHV1-2*02 seqid1=46.3 seqid3=41.5 thrd1=0.257thrd3=0.267 divg=0.182 Nucleic acid sequence=SEQ ID NO: 83 Amino acidsequence=SEQ ID NO: 783; Antibody Heavy Chain No:13817 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094 divg=0.179 Nucleic acidsequence=SEQ ID NO: 84 Amino acid sequence=SEQ ID NO: 784; AntibodyHeavy Chain No:53390 germ=IGHV1-2*02 seqid1=45.5 seqid3=43.8 thrd1=0.177thrd3=0.148 divg=0.179 Nucleic acid sequence=SEQ ID NO: 85 Amino acidsequence=SEQ ID NO: 785; Antibody Heavy Chain No:6665 germ=IGHV1-2*02seqid1=46.3 seqid3=44.6 thrd1=0.178 thrd3=0.150 divg=0.179 Nucleic acidsequence=SEQ ID NO: 86 Amino acid sequence=SEQ ID NO: 786; AntibodyHeavy Chain No:19618 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.192thrd3=0.147 divg=0.179 Nucleic acid sequence=SEQ ID NO: 87 Amino acidsequence=SEQ ID NO: 787; Antibody Heavy Chain No:55471 germ=IGHV1-2*02seqid1=47.9 seqid3=43.8 thrd1=0.204 thrd3=0.208 divg=0.179 Nucleic acidsequence=SEQ ID NO: 88 Amino acid sequence=SEQ ID NO: 788; AntibodyHeavy Chain No:57053 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.180thrd3=0.152 divg=0.176 Nucleic acid sequence=SEQ ID NO: 89 Amino acidsequence=SEQ ID NO: 789; Antibody Heavy Chain No:43247 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.170 thrd3=0.141 divg=0.176 Nucleic acidsequence=SEQ ID NO: 90 Amino acid sequence=SEQ ID NO: 790; AntibodyHeavy Chain No:79236 germ=IGHV1-2*02 seqid1=52.9 seqid3=48.5 thrd1=0.173thrd3=0.145 divg=0.176 Nucleic acid sequence=SEQ ID NO: 91 Amino acidsequence=SEQ ID NO: 791; Antibody Heavy Chain No:72343 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.168 thrd3=0.141 divg=0.176 Nucleic acidsequence=SEQ ID NO: 92 Amino acid sequence=SEQ ID NO: 792; AntibodyHeavy Chain No:100843 germ=IGHV1-2*02 seqid1=49.6 seqid3=45.4thrd1=0.244 thrd3=0.254 divg=0.176 Nucleic acid sequence=SEQ ID NO: 93Amino acid sequence=SEQ ID NO: 793; Antibody Heavy Chain No:26236germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.193 thrd3=0.174divg=0.176 Nucleic acid sequence=SEQ ID NO: 94 Amino acid sequence=SEQID NO: 794; Antibody Heavy Chain No:77123 germ=IGHV1-2*02 seqid1=49.6seqid3=46.2 thrd1=0.190 thrd3=0.176 divg=0.176 Nucleic acid sequence=SEQID NO: 95 Amino acid sequence=SEQ ID NO: 795; Antibody Heavy ChainNo:59418 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.190 thrd3=0.176divg=0.176 Nucleic acid sequence=SEQ ID NO: 96 Amino acid sequence=SEQID NO: 796; Antibody Heavy Chain No:10050 germ=IGHV1-2*02 seqid1=53.7seqid3=49.2 thrd1=0.170 thrd3=0.142 divg=0.172 Nucleic acid sequence=SEQID NO: 97 Amino acid sequence=SEQ ID NO: 797; Antibody Heavy ChainNo:68011 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.191 thrd3=0.172divg=0.172 Nucleic acid sequence=SEQ ID NO: 98 Amino acid sequence=SEQID NO: 798; Antibody Heavy Chain No:72256 germ=IGHV1-2*02 seqid1=39.7seqid3=36.2 thrd1=0.275 thrd3=0.254 divg=0.172 Nucleic acid sequence=SEQID NO: 99 Amino acid sequence=SEQ ID NO: 799; Antibody Heavy ChainNo:42369 germ=IGHV1-2*02 seqid1=39.7 seqid3=38.5 thrd1=0.276 thrd3=0.269divg=0.172 Nucleic acid sequence=SEQ ID NO: 100 Amino acid sequence=SEQID NO: 800; Antibody Heavy Chain No:82140 germ=IGHV1-2*02 seqid1=53.7seqid3=49.2 thrd1=0.147 thrd3=0.094 divg=0.172 Nucleic acid sequence=SEQID NO: 101 Amino acid sequence=SEQ ID NO: 801; Antibody Heavy ChainNo:6646 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094divg=0.172 Nucleic acid sequence=SEQ ID NO: 102 Amino acid sequence=SEQID NO: 802; Antibody Heavy Chain No:62367 germ=IGHV1-2*02 seqid1=49.6seqid3=46.2 thrd1=0.191 thrd3=0.172 divg=0.172 Nucleic acid sequence=SEQID NO: 103 Amino acid sequence=SEQ ID NO: 803; Antibody Heavy ChainNo:84652 germ=IGHV1-2*02 seqid1=51.2 seqid3=47.7 thrd1=0.217 thrd3=0.229divg=0.172 Nucleic acid sequence=SEQ ID NO: 104 Amino acid sequence=SEQID NO: 804; Antibody Heavy Chain No:109302 germ=IGHV1-2*02 seqid1=43.8seqid3=41.5 thrd1=0.257 thrd3=0.241 divg=0.172 Nucleic acid sequence=SEQID NO: 105 Amino acid sequence=SEQ ID NO: 805; Antibody Heavy ChainNo:56047 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.190 thrd3=0.176divg=0.172 Nucleic acid sequence=SEQ ID NO: 106 Amino acid sequence=SEQID NO: 806; Antibody Heavy Chain No:8045 germ=IGHV1-2*02 seqid1=52.9seqid3=48.5 thrd1=0.155 thrd3=0.100 divg=0.172 Nucleic acid sequence=SEQID NO: 107 Amino acid sequence=SEQ ID NO: 807; Antibody Heavy ChainNo:103312 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.191thrd3=0.172 divg=0.172 Nucleic acid sequence=SEQ ID NO: 108 Amino acidsequence=SEQ ID NO: 808; Antibody Heavy Chain No:16454 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.170 thrd3=0.142 divg=0.172 Nucleic acidsequence=SEQ ID NO: 109 Amino acid sequence=SEQ ID NO: 809; AntibodyHeavy Chain No:106554 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2thrd1=0.147 thrd3=0.095 divg=0.172 Nucleic acid sequence=SEQ ID NO: 110Amino acid sequence=SEQ ID NO: 810; Antibody Heavy Chain No:30765germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094divg=0.172 Nucleic acid sequence=SEQ ID NO: 111 Amino acid sequence=SEQID NO: 811; Antibody Heavy Chain No:120328 germ=IGHV1-2*02 seqid1=49.6seqid3=46.2 thrd1=0.169 thrd3=0.099 divg=0.172 Nucleic acid sequence=SEQID NO: 112 Amino acid sequence=SEQ ID NO: 812; Antibody Heavy ChainNo:71818 germ=IGHV1-2*02 seqid1=51.2 seqid3=46.9 thrd1=0.170 thrd3=0.142divg=0.172 Nucleic acid sequence=SEQ ID NO: 113 Amino acid sequence=SEQID NO: 813; Antibody Heavy Chain No:75349 germ=IGHV1-2*02 seqid1=49.6seqid3=46.2 thrd1=0.173 thrd3=0.147 divg=0.172 Nucleic acid sequence=SEQID NO: 114 Amino acid sequence=SEQ ID NO: 814; Antibody Heavy ChainNo:84800 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094divg=0.172 Nucleic acid sequence=SEQ ID NO: 115 Amino acid sequence=SEQID NO: 815; Antibody Heavy Chain No:30534 germ=IGHV1-2*02 seqid1=52.1seqid3=47.7 thrd1=0.277 thrd3=0.171 divg=0.172 Nucleic acid sequence=SEQID NO: 116 Amino acid sequence=SEQ ID NO: 816; Antibody Heavy ChainNo:116747 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.190thrd3=0.176 divg=0.172 Nucleic acid sequence=SEQ ID NO: 117 Amino acidsequence=SEQ ID NO: 817; Antibody Heavy Chain No:59966 germ=IGHV1-2*02seqid1=39.7 seqid3=34.6 thrd1=0.310 thrd3=0.192 divg=0.172 Nucleic acidsequence=SEQ ID NO: 118 Amino acid sequence=SEQ ID NO: 818; AntibodyHeavy Chain No:55155 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.191thrd3=0.172 divg=0.172 Nucleic acid sequence=SEQ ID NO: 119 Amino acidsequence=SEQ ID NO: 819; Antibody Heavy Chain No:44011 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.170 thrd3=0.142 divg=0.172 Nucleic acidsequence=SEQ ID NO: 120 Amino acid sequence=SEQ ID NO: 820; AntibodyHeavy Chain No:52929 germ=IGHV1-2*02 seqid1=48.8 seqid3=45.4 thrd1=0.194thrd3=0.163 divg=0.172 Nucleic acid sequence=SEQ ID NO: 121 Amino acidsequence=SEQ ID NO: 821; Antibody Heavy Chain No:23585 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.170 thrd3=0.142 divg=0.169 Nucleic acidsequence=SEQ ID NO: 122 Amino acid sequence=SEQ ID NO: 822; AntibodyHeavy Chain No:18356 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.177thrd3=0.149 divg=0.169 Nucleic acid sequence=SEQ ID NO: 123 Amino acidsequence=SEQ ID NO: 823; Antibody Heavy Chain No:47548 germ=IGHV1-2*02seqid1=49.6 seqid3=46.2 thrd1=0.190 thrd3=0.176 divg=0.169 Nucleic acidsequence=SEQ ID NO: 124 Amino acid sequence=SEQ ID NO: 824; AntibodyHeavy Chain No:16445 germ=IGHV1-2*02 seqid1=48.8 seqid3=46.2 thrd1=0.178thrd3=0.152 divg=0.169 Nucleic acid sequence=SEQ ID NO: 125 Amino acidsequence=SEQ ID NO: 825; Antibody Heavy Chain No:54787 germ=IGHV1-2*02seqid1=49.6 seqid3=46.2 thrd1=0.173 thrd3=0.147 divg=0.169 Nucleic acidsequence=SEQ ID NO: 126 Amino acid sequence=SEQ ID NO: 826; AntibodyHeavy Chain No:54832 germ=IGHV1-2*02 seqid1=40.5 seqid3=38.5 thrd1=0.217thrd3=0.222 divg=0.169 Nucleic acid sequence=SEQ ID NO: 127 Amino acidsequence=SEQ ID NO: 827; Antibody Heavy Chain No:113458 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.147 thrd3=0.094 divg=0.169 Nucleic acidsequence=SEQ ID NO: 128 Amino acid sequence=SEQ ID NO: 828; AntibodyHeavy Chain No:73745 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.147thrd3=0.094 divg=0.169 Nucleic acid sequence=SEQ ID NO: 129 Amino acidsequence=SEQ ID NO: 829; Antibody Heavy Chain No:63091 germ=IGHV1-2*02seqid1=33.9 seqid3=30.8 thrd1=0.239 thrd3=0.246 divg=0.169 Nucleic acidsequence=SEQ ID NO: 130 Amino acid sequence=SEQ ID NO: 830; AntibodyHeavy Chain No:55725 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.191thrd3=0.172 divg=0.169 Nucleic acid sequence=SEQ ID NO: 131 Amino acidsequence=SEQ ID NO: 831; Antibody Heavy Chain No:107427 germ=IGHV1-2*02seqid1=49.6 seqid3=46.2 thrd1=0.190 thrd3=0.176 divg=0.169 Nucleic acidsequence=SEQ ID NO: 132 Amino acid sequence=SEQ ID NO: 832; AntibodyHeavy Chain No:112250 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2thrd1=0.170 thrd3=0.142 divg=0.169 Nucleic acid sequence=SEQ ID NO: 133Amino acid sequence=SEQ ID NO: 833; Antibody Heavy Chain No:86179germ=IGHV1-2*02 seqid1=49.6 seqid3=46.2 thrd1=0.190 thrd3=0.176divg=0.169 Nucleic acid sequence=SEQ ID NO: 134 Amino acid sequence=SEQID NO: 834; Antibody Heavy Chain No:111621 germ=IGHV1-2*02 seqid1=49.6seqid3=46.2 thrd1=0.172 thrd3=0.149 divg=0.169 Nucleic acid sequence=SEQID NO: 135 Amino acid sequence=SEQ ID NO: 835; Antibody Heavy ChainNo:18139 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.170 thrd3=0.142divg=0.169 Nucleic acid sequence=SEQ ID NO: 136 Amino acid sequence=SEQID NO: 836; Antibody Heavy Chain No:56200 germ=IGHV1-2*02 seqid1=52.9seqid3=50.8 thrd1=0.150 thrd3=0.104 divg=0.166 Nucleic acid sequence=SEQID NO: 137 Amino acid sequence=SEQ ID NO: 837; Antibody Heavy ChainNo:75470 germ=IGHV1-2*02 seqid1=52.9 seqid3=50.8 thrd1=0.150 thrd3=0.104divg=0.166 Nucleic acid sequence=SEQ ID NO: 138 Amino acid sequence=SEQID NO: 838; Antibody Heavy Chain No:48168 germ=IGHV1-2*02 seqid1=47.9seqid3=44.6 thrd1=0.155 thrd3=0.191 divg=0.166 Nucleic acid sequence=SEQID NO: 139 Amino acid sequence=SEQ ID NO: 839; Antibody Heavy ChainNo:78596 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.194 thrd3=0.122divg=0.166 Nucleic acid sequence=SEQ ID NO: 140 Amino acid sequence=SEQID NO: 840; Antibody Heavy Chain No:69129 germ=IGHV1-2*02 seqid1=52.9seqid3=50.8 thrd1=0.150 thrd3=0.104 divg=0.166 Nucleic acid sequence=SEQID NO: 141 Amino acid sequence=SEQ ID NO: 841; Antibody Heavy ChainNo:107212 germ=IGHV1-2*02 seqid1=47.9 seqid3=44.6 thrd1=0.151thrd3=0.189 divg=0.162 Nucleic acid sequence=SEQ ID NO: 142 Amino acidsequence=SEQ ID NO: 842; Antibody Heavy Chain No:71549 germ=IGHV1-2*02seqid1=47.9 seqid3=44.6 thrd1=0.151 thrd3=0.189 divg=0.162 Nucleic acidsequence=SEQ ID NO: 143 Amino acid sequence=SEQ ID NO: 843; AntibodyHeavy Chain No:19124 germ=IGHV1-2*02 seqid1=45.5 seqid3=42.3 thrd1=0.153thrd3=0.196 divg=0.162 Nucleic acid sequence=SEQ ID NO: 144 Amino acidsequence=SEQ ID NO: 844; Antibody Heavy Chain No:100576 germ=IGHV1-2*02seqid1=52.9 seqid3=50.8 thrd1=0.190 thrd3=0.118 divg=0.162 Nucleic acidsequence=SEQ ID NO: 145 Amino acid sequence=SEQ ID NO: 845; AntibodyHeavy Chain No:14535 germ=IGHV1-2*02 seqid1=47.9 seqid3=44.6 thrd1=0.151thrd3=0.189 divg=0.162 Nucleic acid sequence=SEQ ID NO: 146 Amino acidsequence=SEQ ID NO: 846; Antibody Heavy Chain No:11019 germ=IGHV1-2*02seqid1=49.6 seqid3=44.6 thrd1=0.152 thrd3=0.187 divg=0.162 Nucleic acidsequence=SEQ ID NO: 147 Amino acid sequence=SEQ ID NO: 847; AntibodyHeavy Chain No:123937 germ=IGHV1-2*02 seqid1=47.9 seqid3=45.4thrd1=0.218 thrd3=0.219 divg=0.159 Nucleic acid sequence=SEQ ID NO: 148Amino acid sequence=SEQ ID NO: 848; Antibody Heavy Chain No:36310germ=IGHV1-2*02 seqid1=51.2 seqid3=46.9 thrd1=0.161 thrd3=0.236divg=0.159 Nucleic acid sequence=SEQ ID NO: 149 Amino acid sequence=SEQID NO: 849; Antibody Heavy Chain No:32707 germ=IGHV1-2*02 seqid1=47.9seqid3=46.2 thrd1=0.214 thrd3=0.187 divg=0.159 Nucleic acid sequence=SEQID NO: 150 Amino acid sequence=SEQ ID NO: 850; Antibody Heavy ChainNo:87704 germ=IGHV1-2*02 seqid1=47.1 seqid3=43.8 thrd1=0.219 thrd3=0.147divg=0.159 Nucleic acid sequence=SEQ ID NO: 151 Amino acid sequence=SEQID NO: 851; Antibody Heavy Chain No:19098 germ=IGHV1-2*02 seqid1=47.9seqid3=44.6 thrd1=0.151 thrd3=0.189 divg=0.159 Nucleic acid sequence=SEQID NO: 152 Amino acid sequence=SEQ ID NO: 852; Antibody Heavy ChainNo:99181 germ=IGHV1-2*02 seqid1=38.8 seqid3=37.7 thrd1=0.211 thrd3=0.213divg=0.155 Nucleic acid sequence=SEQ ID NO: 153 Amino acid sequence=SEQID NO: 853; Antibody Heavy Chain No:40537 germ=IGHV1-2*02 seqid1=47.9seqid3=46.2 thrd1=0.212 thrd3=0.186 divg=0.155 Nucleic acid sequence=SEQID NO: 154 Amino acid sequence=SEQ ID NO: 854; Antibody Heavy ChainNo:36907 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.150 thrd3=0.149divg=0.155 Nucleic acid sequence=SEQ ID NO: 155 Amino acid sequence=SEQID NO: 855; Antibody Heavy Chain No:29608 germ=IGHV1-2*02 seqid1=47.9seqid3=46.2 thrd1=0.149 thrd3=0.205 divg=0.155 Nucleic acid sequence=SEQID NO: 156 Amino acid sequence=SEQ ID NO: 856; Antibody Heavy ChainNo:113080 germ=IGHV1-2*02 seqid1=48.8 seqid3=48.5 thrd1=0.163thrd3=0.167 divg=0.155 Nucleic acid sequence=SEQ ID NO: 157 Amino acidsequence=SEQ ID NO: 857; Antibody Heavy Chain No:71038 germ=IGHV1-2*02seqid1=47.9 seqid3=46.2 thrd1=0.212 thrd3=0.186 divg=0.155 Nucleic acidsequence=SEQ ID NO: 158 Amino acid sequence=SEQ ID NO: 858; AntibodyHeavy Chain No:6143 germ=IGHV1-2*02 seqid1=52.9 seqid3=48.5 thrd1=0.094thrd3=0.134 divg=0.152 Nucleic acid sequence=SEQ ID NO: 159 Amino acidsequence=SEQ ID NO: 859; Antibody Heavy Chain No:43158 germ=IGHV1-2*02seqid1=47.9 seqid3=46.2 thrd1=0.149 thrd3=0.205 divg=0.152 Nucleic acidsequence=SEQ ID NO: 160 Amino acid sequence=SEQ ID NO: 860; AntibodyHeavy Chain No:15966 germ=IGHV1-2*02 seqid1=52.9 seqid3=45.4 thrd1=0.196thrd3=0.118 divg=0.152 Nucleic acid sequence=SEQ ID NO: 161 Amino acidsequence=SEQ ID NO: 861; Antibody Heavy Chain No:34203 germ=IGHV1-2*02seqid1=49.6 seqid3=45.4 thrd1=0.150 thrd3=0.184 divg=0.152 Nucleic acidsequence=SEQ ID NO: 162 Amino acid sequence=SEQ ID NO: 862; AntibodyHeavy Chain No:88799 germ=IGHV1-2*02 seqid1=46.3 seqid3=42.3 thrd1=0.168thrd3=0.213 divg=0.152 Nucleic acid sequence=SEQ ID NO: 163 Amino acidsequence=SEQ ID NO: 863; Antibody Heavy Chain No:2164 germ=IGHV1-2*02seqid1=51.2 seqid3=50.0 thrd1=0.150 thrd3=0.150 divg=0.152 Nucleic acidsequence=SEQ ID NO: 164 Amino acid sequence=SEQ ID NO: 864; AntibodyHeavy Chain No:179552 germ=IGHV1-2*02 seqid1=43.8 seqid3=45.4thrd1=0.216 thrd3=0.125 divg=0.149 Nucleic acid sequence=SEQ ID NO: 165Amino acid sequence=SEQ ID NO: 865; Antibody Heavy Chain No:4734germ=IGHV1-2*02 seqid1=44.6 seqid3=48.5 thrd1=0.185 thrd3=0.156divg=0.145 Nucleic acid sequence=SEQ ID NO: 166 Amino acid sequence=SEQID NO: 866; Antibody Heavy Chain No:17242 germ=IGHV1-2*02 seqid1=47.9seqid3=46.2 thrd1=0.144 thrd3=0.143 divg=0.145 Nucleic acid sequence=SEQID NO: 167 Amino acid sequence=SEQ ID NO: 867; Antibody Heavy ChainNo:17884 germ=IGHV1-2*02 seqid1=44.6 seqid3=48.5 thrd1=0.186 thrd3=0.158divg=0.142 Nucleic acid sequence=SEQ ID NO: 168 Amino acid sequence=SEQID NO: 868; Antibody Heavy Chain No:32382 germ=IGHV1-2*02 seqid1=49.6seqid3=47.7 thrd1=0.144 thrd3=0.143 divg=0.142 Nucleic acid sequence=SEQID NO: 169 Amino acid sequence=SEQ ID NO: 869; Antibody Heavy ChainNo:41962 germ=IGHV1-2*02 seqid1=49.6 seqid3=47.7 thrd1=0.144 thrd3=0.143divg=0.142 Nucleic acid sequence=SEQ ID NO: 170 Amino acid sequence=SEQID NO: 870; Antibody Heavy Chain No:59875 germ=IGHV1-2*02 seqid1=49.6seqid3=47.7 thrd1=0.144 thrd3=0.143 divg=0.142 Nucleic acid sequence=SEQID NO: 171 Amino acid sequence=SEQ ID NO: 871; Antibody Heavy ChainNo:33133 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.163 thrd3=0.158divg=0.142 Nucleic acid sequence=SEQ ID NO: 172 Amino acid sequence=SEQID NO: 872; Antibody Heavy Chain No:82539 germ=IGHV1-2*02 seqid1=53.7seqid3=48.5 thrd1=0.173 thrd3=0.204 divg=0.142 Nucleic acid sequence=SEQID NO: 173 Amino acid sequence=SEQ ID NO: 873; Antibody Heavy ChainNo:43232 germ=IGHV1-2*02 seqid1=49.6 seqid3=47.7 thrd1=0.144 thrd3=0.143divg=0.142 Nucleic acid sequence=SEQ ID NO: 174 Amino acid sequence=SEQID NO: 874; Antibody Heavy Chain No:49896 germ=IGHV1-2*02 seqid1=43.0seqid3=43.8 thrd1=0.240 thrd3=0.155 divg=0.142 Nucleic acid sequence=SEQID NO: 175 Amino acid sequence=SEQ ID NO: 875; Antibody Heavy ChainNo:13144 germ=IGHV1-2*02 seqid1=53.7 seqid3=48.5 thrd1=0.208 thrd3=0.114divg=0.139 Nucleic acid sequence=SEQ ID NO: 176 Amino acid sequence=SEQID NO: 876; Antibody Heavy Chain No:94912 germ=IGHV1-2*02 seqid1=52.1seqid3=49.2 thrd1=0.163 thrd3=0.210 divg=0.139 Nucleic acid sequence=SEQID NO: 177 Amino acid sequence=SEQ ID NO: 877; Antibody Heavy ChainNo:95649 germ=IGHV1-2*02 seqid1=51.2 seqid3=45.4 thrd1=0.106 thrd3=0.141divg=0.139 Nucleic acid sequence=SEQ ID NO: 178 Amino acid sequence=SEQID NO: 878; Antibody Heavy Chain No:5273 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.139 Nucleic acid sequence=SEQID NO: 179 Amino acid sequence=SEQ ID NO: 879; Antibody Heavy ChainNo:79693 germ=IGHV1-2*02 seqid1=52.1 seqid3=46.2 thrd1=0.092 thrd3=0.132divg=0.139 Nucleic acid sequence=SEQ ID NO: 180 Amino acid sequence=SEQID NO: 880; Antibody Heavy Chain No:80554 germ=IGHV1-2*02 seqid1=51.2seqid3=48.5 thrd1=0.161 thrd3=0.211 divg=0.139 Nucleic acid sequence=SEQID NO: 181 Amino acid sequence=SEQ ID NO: 881; Antibody Heavy ChainNo:119108 germ=IGHV1-2*02 seqid1=47.1 seqid3=45.4 thrd1=0.163thrd3=0.222 divg=0.139 Nucleic acid sequence=SEQ ID NO: 182 Amino acidsequence=SEQ ID NO: 882; Antibody Heavy Chain No:29950 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.139 Nucleic acidsequence=SEQ ID NO: 183 Amino acid sequence=SEQ ID NO: 883; AntibodyHeavy Chain No:39089 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.139 Nucleic acid sequence=SEQ ID NO: 184 Amino acidsequence=SEQ ID NO: 884; Antibody Heavy Chain No:40410 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.160 thrd3=0.155 divg=0.139 Nucleic acidsequence=SEQ ID NO: 185 Amino acid sequence=SEQ ID NO: 885; AntibodyHeavy Chain No:14930 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.161thrd3=0.211 divg=0.139 Nucleic acid sequence=SEQ ID NO: 186 Amino acidsequence=SEQ ID NO: 886; Antibody Heavy Chain No:55973 germ=IGHV1-2*02seqid1=49.6 seqid3=48.5 thrd1=0.170 thrd3=0.115 divg=0.139 Nucleic acidsequence=SEQ ID NO: 187 Amino acid sequence=SEQ ID NO: 887; AntibodyHeavy Chain No:4040 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.163thrd3=0.159 divg=0.139 Nucleic acid sequence=SEQ ID NO: 188 Amino acidsequence=SEQ ID NO: 888; Antibody Heavy Chain No:52215 germ=IGHV1-2*02seqid1=44.6 seqid3=44.6 thrd1=0.189 thrd3=0.284 divg=0.139 Nucleic acidsequence=SEQ ID NO: 189 Amino acid sequence=SEQ ID NO: 889; AntibodyHeavy Chain No:65622 germ=IGHV1-2*02 seqid1=57.9 seqid3=51.5 thrd1=0.228thrd3=0.174 divg=0.139 Nucleic acid sequence=SEQ ID NO: 190 Amino acidsequence=SEQ ID NO: 890; Antibody Heavy Chain No:144394 germ=IGHV1-2*02seqid1=50.4 seqid3=48.5 thrd1=0.148 thrd3=0.151 divg=0.139 Nucleic acidsequence=SEQ ID NO: 191 Amino acid sequence=SEQ ID NO: 891; AntibodyHeavy Chain No:10658 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.139 Nucleic acid sequence=SEQ ID NO: 192 Amino acidsequence=SEQ ID NO: 892; Antibody Heavy Chain No:3811 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.139 Nucleic acidsequence=SEQ ID NO: 193 Amino acid sequence=SEQ ID NO: 893; AntibodyHeavy Chain No:48101 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.162thrd3=0.157 divg=0.139 Nucleic acid sequence=SEQ ID NO: 194 Amino acidsequence=SEQ ID NO: 894; Antibody Heavy Chain No:15636 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.139 Nucleic acidsequence=SEQ ID NO: 195 Amino acid sequence=SEQ ID NO: 895; AntibodyHeavy Chain No:26505 germ=IGHV1-2*02 seqid1=48.8 seqid3=43.8 thrd1=0.283thrd3=0.157 divg=0.139 Nucleic acid sequence=SEQ ID NO: 196 Amino acidsequence=SEQ ID NO: 896; Antibody Heavy Chain No:84089 germ=IGHV1-2*02seqid1=51.2 seqid3=50.0 thrd1=0.159 thrd3=0.154 divg=0.135 Nucleic acidsequence=SEQ ID NO: 197 Amino acid sequence=SEQ ID NO: 897; AntibodyHeavy Chain No:104154 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.135 Nucleic acid sequence=SEQ ID NO: 198Amino acid sequence=SEQ ID NO: 898; Antibody Heavy Chain No:23977germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.159 thrd3=0.155divg=0.135 Nucleic acid sequence=SEQ ID NO: 199 Amino acid sequence=SEQID NO: 899; Antibody Heavy Chain No:167681 germ=IGHV1-2*02 seqid1=41.3seqid3=40.0 thrd1=0.234 thrd3=0.259 divg=0.135 Nucleic acid sequence=SEQID NO: 200 Amino acid sequence=SEQ ID NO: 900; Antibody Heavy ChainNo:17288 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.135 Nucleic acid sequence=SEQ ID NO: 201 Amino acid sequence=SEQID NO: 901; Antibody Heavy Chain No:33637 germ=IGHV1-2*02 seqid1=52.9seqid3=47.7 thrd1=0.094 thrd3=0.134 divg=0.135 Nucleic acid sequence=SEQID NO: 202 Amino acid sequence=SEQ ID NO: 902; Antibody Heavy ChainNo:110259 germ=IGHV1-2*02 seqid1=48.8 seqid3=50.0 thrd1=0.161thrd3=0.158 divg=0.135 Nucleic acid sequence=SEQ ID NO: 203 Amino acidsequence=SEQ ID NO: 903; Antibody Heavy Chain No:11461 germ=IGHV1-2*02seqid1=49.6 seqid3=49.2 thrd1=0.164 thrd3=0.160 divg=0.135 Nucleic acidsequence=SEQ ID NO: 204 Amino acid sequence=SEQ ID NO: 904; AntibodyHeavy Chain No:57309 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.164thrd3=0.159 divg=0.135 Nucleic acid sequence=SEQ ID NO: 205 Amino acidsequence=SEQ ID NO: 905; Antibody Heavy Chain No:28613 germ=IGHV1-2*02seqid1=47.1 seqid3=46.9 thrd1=0.180 thrd3=0.242 divg=0.135 Nucleic acidsequence=SEQ ID NO: 206 Amino acid sequence=SEQ ID NO: 906; AntibodyHeavy Chain No:80786 germ=IGHV1-2*02 seqid1=47.1 seqid3=44.6 thrd1=0.159thrd3=0.152 divg=0.135 Nucleic acid sequence=SEQ ID NO: 207 Amino acidsequence=SEQ ID NO: 907; Antibody Heavy Chain No:34770 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.162 thrd3=0.158 divg=0.135 Nucleic acidsequence=SEQ ID NO: 208 Amino acid sequence=SEQ ID NO: 908; AntibodyHeavy Chain No:58396 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.159thrd3=0.155 divg=0.135 Nucleic acid sequence=SEQ ID NO: 209 Amino acidsequence=SEQ ID NO: 909; Antibody Heavy Chain No:150271 germ=IGHV1-2*02seqid1=39.7 seqid3=41.5 thrd1=0.310 thrd3=0.356 divg=0.135 Nucleic acidsequence=SEQ ID NO: 210 Amino acid sequence=SEQ ID NO: 910; AntibodyHeavy Chain No:44015 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.160thrd3=0.157 divg=0.135 Nucleic acid sequence=SEQ ID NO: 211 Amino acidsequence=SEQ ID NO: 911; Antibody Heavy Chain No:64391 germ=IGHV1-2*02seqid1=45.5 seqid3=45.4 thrd1=0.228 thrd3=0.145 divg=0.135 Nucleic acidsequence=SEQ ID NO: 212 Amino acid sequence=SEQ ID NO: 912; AntibodyHeavy Chain No:25120 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.161thrd3=0.157 divg=0.135 Nucleic acid sequence=SEQ ID NO: 213 Amino acidsequence=SEQ ID NO: 913; Antibody Heavy Chain No:10614 germ=IGHV1-2*02seqid1=50.4 seqid3=50.8 thrd1=0.165 thrd3=0.150 divg=0.135 Nucleic acidsequence=SEQ ID NO: 214 Amino acid sequence=SEQ ID NO: 914; AntibodyHeavy Chain No:51689 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.162thrd3=0.158 divg=0.135 Nucleic acid sequence=SEQ ID NO: 215 Amino acidsequence=SEQ ID NO: 915; Antibody Heavy Chain No:63801 germ=IGHV1-2*02seqid1=53.7 seqid3=50.0 thrd1=0.153 thrd3=0.200 divg=0.135 Nucleic acidsequence=SEQ ID NO: 216 Amino acid sequence=SEQ ID NO: 916; AntibodyHeavy Chain No:116007 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7thrd1=0.093 thrd3=0.133 divg=0.135 Nucleic acid sequence=SEQ ID NO: 217Amino acid sequence=SEQ ID NO: 917; Antibody Heavy Chain No:22483germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.164 thrd3=0.159divg=0.135 Nucleic acid sequence=SEQ ID NO: 218 Amino acid sequence=SEQID NO: 918; Antibody Heavy Chain No:52322 germ=IGHV1-2*02 seqid1=52.1seqid3=46.2 thrd1=0.099 thrd3=0.141 divg=0.135 Nucleic acid sequence=SEQID NO: 219 Amino acid sequence=SEQ ID NO: 919; Antibody Heavy ChainNo:88747 germ=IGHV1-2*02 seqid1=47.1 seqid3=46.9 thrd1=0.165 thrd3=0.160divg=0.135 Nucleic acid sequence=SEQ ID NO: 220 Amino acid sequence=SEQID NO: 920; Antibody Heavy Chain No:35449 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.135 divg=0.135 Nucleic acid sequence=SEQID NO: 221 Amino acid sequence=SEQ ID NO: 921; Antibody Heavy ChainNo:18812 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.157 thrd3=0.153divg=0.135 Nucleic acid sequence=SEQ ID NO: 222 Amino acid sequence=SEQID NO: 922; Antibody Heavy Chain No:69878 germ=IGHV1-2*02 seqid1=49.6seqid3=50.0 thrd1=0.149 thrd3=0.121 divg=0.135 Nucleic acid sequence=SEQID NO: 223 Amino acid sequence=SEQ ID NO: 923; Antibody Heavy ChainNo:78844 germ=IGHV1-2*02 seqid1=50.4 seqid3=47.7 thrd1=0.150 thrd3=0.143divg=0.135 Nucleic acid sequence=SEQ ID NO: 224 Amino acid sequence=SEQID NO: 924; Antibody Heavy Chain No:79855 germ=IGHV1-2*02 seqid1=43.0seqid3=43.8 thrd1=0.262 thrd3=0.267 divg=0.135 Nucleic acid sequence=SEQID NO: 225 Amino acid sequence=SEQ ID NO: 925; Antibody Heavy ChainNo:59636 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.162 thrd3=0.158divg=0.135 Nucleic acid sequence=SEQ ID NO: 226 Amino acid sequence=SEQID NO: 926; Antibody Heavy Chain No:81201 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.157 divg=0.135 Nucleic acid sequence=SEQID NO: 227 Amino acid sequence=SEQ ID NO: 927; Antibody Heavy ChainNo:72731 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.160 thrd3=0.155divg=0.135 Nucleic acid sequence=SEQ ID NO: 228 Amino acid sequence=SEQID NO: 928; Antibody Heavy Chain No:195439 germ=IGHV1-2*02 seqid1=49.6seqid3=45.4 thrd1=0.123 thrd3=0.107 divg=0.135 Nucleic acid sequence=SEQID NO: 229 Amino acid sequence=SEQ ID NO: 929; Antibody Heavy ChainNo:52914 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.282 thrd3=0.176divg=0.135 Nucleic acid sequence=SEQ ID NO: 230 Amino acid sequence=SEQID NO: 930; Antibody Heavy Chain No:74803 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.135 Nucleic acid sequence=SEQID NO: 231 Amino acid sequence=SEQ ID NO: 931; Antibody Heavy ChainNo:74459 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.8 thrd1=0.160 thrd3=0.155divg=0.135 Nucleic acid sequence=SEQ ID NO: 232 Amino acid sequence=SEQID NO: 932; Antibody Heavy Chain No:7871 germ=IGHV1-2*02 seqid1=52.1seqid3=47.7 thrd1=0.201 thrd3=0.127 divg=0.135 Nucleic acid sequence=SEQID NO: 233 Amino acid sequence=SEQ ID NO: 933; Antibody Heavy ChainNo:20841 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.135 Nucleic acid sequence=SEQ ID NO: 234 Amino acid sequence=SEQID NO: 934; Antibody Heavy Chain No:37716 germ=IGHV1-2*02 seqid1=49.6seqid3=49.2 thrd1=0.160 thrd3=0.156 divg=0.135 Nucleic acid sequence=SEQID NO: 235 Amino acid sequence=SEQ ID NO: 935; Antibody Heavy ChainNo:39615 germ=IGHV1-2*02 seqid1=50.4 seqid3=47.7 thrd1=0.150 thrd3=0.143divg=0.135 Nucleic acid sequence=SEQ ID NO: 236 Amino acid sequence=SEQID NO: 936; Antibody Heavy Chain No:66215 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.160 thrd3=0.155 divg=0.135 Nucleic acid sequence=SEQID NO: 237 Amino acid sequence=SEQ ID NO: 937; Antibody Heavy ChainNo:82019 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.165 thrd3=0.163divg=0.135 Nucleic acid sequence=SEQ ID NO: 238 Amino acid sequence=SEQID NO: 938; Antibody Heavy Chain No:21464 germ=IGHV1-2*02 seqid1=51.2seqid3=50.0 thrd1=0.162 thrd3=0.156 divg=0.135 Nucleic acid sequence=SEQID NO: 239 Amino acid sequence=SEQ ID NO: 939; Antibody Heavy ChainNo:105467 germ=IGHV1-2*02 seqid1=49.6 seqid3=47.7 thrd1=0.167thrd3=0.164 divg=0.135 Nucleic acid sequence=SEQ ID NO: 240 Amino acidsequence=SEQ ID NO: 940; Antibody Heavy Chain No:60368 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.135 Nucleic acidsequence=SEQ ID NO: 241 Amino acid sequence=SEQ ID NO: 941; AntibodyHeavy Chain No:113424 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 242Amino acid sequence=SEQ ID NO: 942; Antibody Heavy Chain No:87796germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 243 Amino acid sequence=SEQID NO: 943; Antibody Heavy Chain No:107864 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 244 Amino acid sequence=SEQ ID NO: 944; Antibody Heavy ChainNo:128805 germ=IGHV1-2*02 seqid1=48.8 seqid3=47.7 thrd1=0.153thrd3=0.162 divg=0.132 Nucleic acid sequence=SEQ ID NO: 245 Amino acidsequence=SEQ ID NO: 945; Antibody Heavy Chain No:38060 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 246 Amino acid sequence=SEQ ID NO: 946; AntibodyHeavy Chain No:97440 germ=IGHV1-2*02 seqid1=48.8 seqid3=48.5 thrd1=0.165thrd3=0.162 divg=0.132 Nucleic acid sequence=SEQ ID NO: 247 Amino acidsequence=SEQ ID NO: 947; Antibody Heavy Chain No:66266 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 248 Amino acid sequence=SEQ ID NO: 948; AntibodyHeavy Chain No:39042 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.159thrd3=0.138 divg=0.132 Nucleic acid sequence=SEQ ID NO: 249 Amino acidsequence=SEQ ID NO: 949; Antibody Heavy Chain No:94534 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 250 Amino acid sequence=SEQ ID NO: 950; AntibodyHeavy Chain No:94385 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 251 Amino acidsequence=SEQ ID NO: 951; Antibody Heavy Chain No:76265 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 252 Amino acid sequence=SEQ ID NO: 952; AntibodyHeavy Chain No:22535 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 253 Amino acidsequence=SEQ ID NO: 953; Antibody Heavy Chain No:85110 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 254 Amino acid sequence=SEQ ID NO: 954; AntibodyHeavy Chain No:168379 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2thrd1=0.162 thrd3=0.157 divg=0.132 Nucleic acid sequence=SEQ ID NO: 255Amino acid sequence=SEQ ID NO: 955; Antibody Heavy Chain No:112172germ=IGHV1-2*02 seqid1=50.4 seqid3=44.6 thrd1=0.105 thrd3=0.149divg=0.132 Nucleic acid sequence=SEQ ID NO: 256 Amino acid sequence=SEQID NO: 956; Antibody Heavy Chain No:47329 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 257 Amino acid sequence=SEQ ID NO: 957; Antibody Heavy ChainNo:38803 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 258 Amino acid sequence=SEQID NO: 958; Antibody Heavy Chain No:42945 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 259 Amino acid sequence=SEQ ID NO: 959; Antibody Heavy ChainNo:55338 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 260 Amino acid sequence=SEQID NO: 960; Antibody Heavy Chain No:53837 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQID NO: 261 Amino acid sequence=SEQ ID NO: 961; Antibody Heavy ChainNo:73759 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 262 Amino acid sequence=SEQID NO: 962; Antibody Heavy Chain No:131368 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 263 Amino acid sequence=SEQ ID NO: 963; Antibody Heavy ChainNo:80330 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 264 Amino acid sequence=SEQID NO: 964; Antibody Heavy Chain No:52164 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 265 Amino acid sequence=SEQ ID NO: 965; Antibody Heavy ChainNo:52134 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 266 Amino acid sequence=SEQID NO: 966; Antibody Heavy Chain No:53605 germ=IGHV1-2*02 seqid1=51.2seqid3=46.9 thrd1=0.172 thrd3=0.222 divg=0.132 Nucleic acid sequence=SEQID NO: 267 Amino acid sequence=SEQ ID NO: 967; Antibody Heavy ChainNo:129544 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQ ID NO: 268 Amino acidsequence=SEQ ID NO: 968; Antibody Heavy Chain No:28518 germ=IGHV1-2*02seqid1=54.5 seqid3=50.0 thrd1=0.179 thrd3=0.175 divg=0.132 Nucleic acidsequence=SEQ ID NO: 269 Amino acid sequence=SEQ ID NO: 969; AntibodyHeavy Chain No:38525 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 270 Amino acidsequence=SEQ ID NO: 970; Antibody Heavy Chain No:88750 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 271 Amino acid sequence=SEQ ID NO: 971; AntibodyHeavy Chain No:26549 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2 thrd1=0.159thrd3=0.175 divg=0.132 Nucleic acid sequence=SEQ ID NO: 272 Amino acidsequence=SEQ ID NO: 972; Antibody Heavy Chain No:7631 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 273 Amino acid sequence=SEQ ID NO: 973; AntibodyHeavy Chain No:130129 germ=IGHV1-2*02 seqid1=48.8 seqid3=49.2thrd1=0.159 thrd3=0.153 divg=0.132 Nucleic acid sequence=SEQ ID NO: 274Amino acid sequence=SEQ ID NO: 974; Antibody Heavy Chain No:16331germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 275 Amino acid sequence=SEQID NO: 975; Antibody Heavy Chain No:60175 germ=IGHV1-2*02 seqid1=50.4seqid3=49.2 thrd1=0.160 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 276 Amino acid sequence=SEQ ID NO: 976; Antibody Heavy ChainNo:171282 germ=IGHV1-2*02 seqid1=51.2 seqid3=51.5 thrd1=0.153thrd3=0.192 divg=0.132 Nucleic acid sequence=SEQ ID NO: 277 Amino acidsequence=SEQ ID NO: 977; Antibody Heavy Chain No:63220 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 278 Amino acid sequence=SEQ ID NO: 978; AntibodyHeavy Chain No:53630 germ=IGHV1-2*02 seqid1=50.4 seqid3=47.7 thrd1=0.232thrd3=0.169 divg=0.132 Nucleic acid sequence=SEQ ID NO: 279 Amino acidsequence=SEQ ID NO: 979; Antibody Heavy Chain No:31451 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 280 Amino acid sequence=SEQ ID NO: 980; AntibodyHeavy Chain No:40126 germ=IGHV1-2*02 seqid1=49.6 seqid3=48.5 thrd1=0.166thrd3=0.164 divg=0.132 Nucleic acid sequence=SEQ ID NO: 281 Amino acidsequence=SEQ ID NO: 981; Antibody Heavy Chain No:66324 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 282 Amino acid sequence=SEQ ID NO: 982; AntibodyHeavy Chain No:13887 germ=IGHV1-2*02 seqid1=43.0 seqid3=44.6 thrd1=0.258thrd3=0.192 divg=0.132 Nucleic acid sequence=SEQ ID NO: 283 Amino acidsequence=SEQ ID NO: 983; Antibody Heavy Chain No:27307 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 284 Amino acid sequence=SEQ ID NO: 984; AntibodyHeavy Chain No:26779 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 285 Amino acidsequence=SEQ ID NO: 985; Antibody Heavy Chain No:171898 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 286 Amino acid sequence=SEQ ID NO: 986; AntibodyHeavy Chain No:13137 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 287 Amino acidsequence=SEQ ID NO: 987; Antibody Heavy Chain No:87705 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 288 Amino acid sequence=SEQ ID NO: 988; AntibodyHeavy Chain No:113257 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 289Amino acid sequence=SEQ ID NO: 989; Antibody Heavy Chain No:29451germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 290 Amino acid sequence=SEQID NO: 990; Antibody Heavy Chain No:163834 germ=IGHV1-2*02 seqid1=47.1seqid3=45.4 thrd1=0.188 thrd3=0.188 divg=0.132 Nucleic acid sequence=SEQID NO: 291 Amino acid sequence=SEQ ID NO: 991; Antibody Heavy ChainNo:99313 germ=IGHV1-2*02 seqid1=49.6 seqid3=43.1 thrd1=0.099 thrd3=0.136divg=0.132 Nucleic acid sequence=SEQ ID NO: 292 Amino acid sequence=SEQID NO: 992; Antibody Heavy Chain No:94039 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 293 Amino acid sequence=SEQ ID NO: 993; Antibody Heavy ChainNo:44862 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 294 Amino acid sequence=SEQID NO: 994; Antibody Heavy Chain No:90451 germ=IGHV1-2*02 seqid1=49.6seqid3=49.2 thrd1=0.160 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 295 Amino acid sequence=SEQ ID NO: 995; Antibody Heavy ChainNo:62365 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 296 Amino acid sequence=SEQID NO: 996; Antibody Heavy Chain No:137307 germ=IGHV1-2*02 seqid1=40.5seqid3=41.5 thrd1=0.187 thrd3=0.183 divg=0.132 Nucleic acid sequence=SEQID NO: 297 Amino acid sequence=SEQ ID NO: 997; Antibody Heavy ChainNo:35237 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.132 Nucleic acid sequence=SEQ ID NO: 298 Amino acid sequence=SEQID NO: 998; Antibody Heavy Chain No:85861 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 299 Amino acid sequence=SEQ ID NO: 999; Antibody Heavy ChainNo:44765 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 300 Amino acid sequence=SEQID NO: 1000; Antibody Heavy Chain No:64006 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 301 Amino acid sequence=SEQ ID NO: 1001; Antibody Heavy ChainNo:30706 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 302 Amino acid sequence=SEQID NO: 1002; Antibody Heavy Chain No:66921 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 303 Amino acid sequence=SEQ ID NO: 1003; Antibody Heavy ChainNo:56340 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 304 Amino acid sequence=SEQID NO: 1004; Antibody Heavy Chain No:54385 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 305 Amino acid sequence=SEQ ID NO: 1005; Antibody Heavy ChainNo:107464 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 306 Amino acidsequence=SEQ ID NO: 1006; Antibody Heavy Chain No:72997 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 307 Amino acid sequence=SEQ ID NO: 1007; AntibodyHeavy Chain No:122440 germ=IGHV1-2*02 seqid1=48.8 seqid3=48.5thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 308Amino acid sequence=SEQ ID NO: 1008; Antibody Heavy Chain No:51611germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 309 Amino acid sequence=SEQID NO: 1009; Antibody Heavy Chain No:47198 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 310 Amino acid sequence=SEQ ID NO: 1010; Antibody Heavy ChainNo:90259 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.132 Nucleic acid sequence=SEQ ID NO: 311 Amino acid sequence=SEQID NO: 1011; Antibody Heavy Chain No:81876 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 312 Amino acid sequence=SEQ ID NO: 1012; Antibody Heavy ChainNo:89609 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 313 Amino acid sequence=SEQID NO: 1013; Antibody Heavy Chain No:35904 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.096 thrd3=0.137 divg=0.132 Nucleic acid sequence=SEQID NO: 314 Amino acid sequence=SEQ ID NO: 1014; Antibody Heavy ChainNo:111059 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 315 Amino acidsequence=SEQ ID NO: 1015; Antibody Heavy Chain No:16161 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 316 Amino acid sequence=SEQ ID NO: 1016; AntibodyHeavy Chain No:110880 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQ ID NO: 317Amino acid sequence=SEQ ID NO: 1017; Antibody Heavy Chain No:14765germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 318 Amino acid sequence=SEQID NO: 1018; Antibody Heavy Chain No:84533 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 319 Amino acid sequence=SEQ ID NO: 1019; Antibody Heavy ChainNo:66611 germ=IGHV1-2*02 seqid1=48.8 seqid3=47.7 thrd1=0.153 thrd3=0.162divg=0.132 Nucleic acid sequence=SEQ ID NO: 320 Amino acid sequence=SEQID NO: 1020; Antibody Heavy Chain No:8697 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQID NO: 321 Amino acid sequence=SEQ ID NO: 1021; Antibody Heavy ChainNo:164229 germ=IGHV1-2*02 seqid1=50.4 seqid3=43.8 thrd1=0.171thrd3=0.168 divg=0.132 Nucleic acid sequence=SEQ ID NO: 322 Amino acidsequence=SEQ ID NO: 1022; Antibody Heavy Chain No:31295 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 323 Amino acid sequence=SEQ ID NO: 1023; AntibodyHeavy Chain No:25528 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 324 Amino acidsequence=SEQ ID NO: 1024; Antibody Heavy Chain No:83584 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 325 Amino acid sequence=SEQ ID NO: 1025; AntibodyHeavy Chain No:40574 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 326 Amino acidsequence=SEQ ID NO: 1026; Antibody Heavy Chain No:118566 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 327 Amino acid sequence=SEQ ID NO: 1027; AntibodyHeavy Chain No:64619 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQ ID NO: 328 Amino acidsequence=SEQ ID NO: 1028; Antibody Heavy Chain No:112548 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 329 Amino acid sequence=SEQ ID NO: 1029; AntibodyHeavy Chain No:80024 germ=IGHV1-2*02 seqid1=47.9 seqid3=47.7 thrd1=0.160thrd3=0.155 divg=0.132 Nucleic acid sequence=SEQ ID NO: 330 Amino acidsequence=SEQ ID NO: 1030; Antibody Heavy Chain No:53014 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 331 Amino acid sequence=SEQ ID NO: 1031; AntibodyHeavy Chain No:22889 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 332 Amino acidsequence=SEQ ID NO: 1032; Antibody Heavy Chain No:20603 germ=IGHV1-2*02seqid1=52.1 seqid3=46.2 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 333 Amino acid sequence=SEQ ID NO: 1033; AntibodyHeavy Chain No:95127 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 334 Amino acidsequence=SEQ ID NO: 1034; Antibody Heavy Chain No:40882 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 335 Amino acid sequence=SEQ ID NO: 1035; AntibodyHeavy Chain No:12779 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 336 Amino acidsequence=SEQ ID NO: 1036; Antibody Heavy Chain No:30393 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 337 Amino acid sequence=SEQ ID NO: 1037; AntibodyHeavy Chain No:44033 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 338 Amino acidsequence=SEQ ID NO: 1038; Antibody Heavy Chain No:13199 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 339 Amino acid sequence=SEQ ID NO: 1039; AntibodyHeavy Chain No:32566 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 340 Amino acidsequence=SEQ ID NO: 1040; Antibody Heavy Chain No:64977 germ=IGHV1-2*02seqid1=48.8 seqid3=47.7 thrd1=0.153 thrd3=0.162 divg=0.132 Nucleic acidsequence=SEQ ID NO: 341 Amino acid sequence=SEQ ID NO: 1041; AntibodyHeavy Chain No:83943 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 342 Amino acidsequence=SEQ ID NO: 1042; Antibody Heavy Chain No:93877 germ=IGHV1-2*02seqid1=50.4 seqid3=44.6 thrd1=0.125 thrd3=0.189 divg=0.132 Nucleic acidsequence=SEQ ID NO: 343 Amino acid sequence=SEQ ID NO: 1043; AntibodyHeavy Chain No:18689 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 344 Amino acidsequence=SEQ ID NO: 1044; Antibody Heavy Chain No:29339 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 345 Amino acid sequence=SEQ ID NO: 1045; AntibodyHeavy Chain No:63209 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQ ID NO: 346 Amino acidsequence=SEQ ID NO: 1046; Antibody Heavy Chain No:78362 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 347 Amino acid sequence=SEQ ID NO: 1047; AntibodyHeavy Chain No:83864 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQ ID NO: 348 Amino acidsequence=SEQ ID NO: 1048; Antibody Heavy Chain No:39670 germ=IGHV1-2*02seqid1=48.8 seqid3=48.5 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 349 Amino acid sequence=SEQ ID NO: 1049; AntibodyHeavy Chain No:113372 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 350Amino acid sequence=SEQ ID NO: 1050; Antibody Heavy Chain No:55937germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 351 Amino acid sequence=SEQID NO: 1051; Antibody Heavy Chain No:30882 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 352 Amino acid sequence=SEQ ID NO: 1052; Antibody Heavy ChainNo:121824 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 353 Amino acidsequence=SEQ ID NO: 1053; Antibody Heavy Chain No:36567 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 354 Amino acid sequence=SEQ ID NO: 1054; AntibodyHeavy Chain No:112475 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 355Amino acid sequence=SEQ ID NO: 1055; Antibody Heavy Chain No:38636germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 356 Amino acid sequence=SEQID NO: 1056; Antibody Heavy Chain No:47990 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 357 Amino acid sequence=SEQ ID NO: 1057; Antibody Heavy ChainNo:69898 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 358 Amino acid sequence=SEQID NO: 1058; Antibody Heavy Chain No:22432 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 359 Amino acid sequence=SEQ ID NO: 1059; Antibody Heavy ChainNo:51559 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 360 Amino acid sequence=SEQID NO: 1060; Antibody Heavy Chain No:104370 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 361 Amino acid sequence=SEQ ID NO: 1061; Antibody Heavy ChainNo:70186 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 362 Amino acid sequence=SEQID NO: 1062; Antibody Heavy Chain No:66807 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 363 Amino acid sequence=SEQ ID NO: 1063; Antibody Heavy ChainNo:36621 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 364 Amino acid sequence=SEQID NO: 1064; Antibody Heavy Chain No:16321 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 365 Amino acid sequence=SEQ ID NO: 1065; Antibody Heavy ChainNo:50350 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 366 Amino acid sequence=SEQID NO: 1066; Antibody Heavy Chain No:182546 germ=IGHV1-2*02 seqid1=46.3seqid3=43.1 thrd1=0.179 thrd3=0.143 divg=0.132 Nucleic acid sequence=SEQID NO: 367 Amino acid sequence=SEQ ID NO: 1067; Antibody Heavy ChainNo:87519 germ=IGHV1-2*02 seqid1=47.9 seqid3=47.7 thrd1=0.162 thrd3=0.157divg=0.132 Nucleic acid sequence=SEQ ID NO: 368 Amino acid sequence=SEQID NO: 1068; Antibody Heavy Chain No:38456 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 369 Amino acid sequence=SEQ ID NO: 1069; Antibody Heavy ChainNo:92098 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.132 Nucleic acid sequence=SEQ ID NO: 370 Amino acid sequence=SEQID NO: 1070; Antibody Heavy Chain No:98786 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 371 Amino acid sequence=SEQ ID NO: 1071; Antibody Heavy ChainNo:79026 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.163 thrd3=0.157divg=0.132 Nucleic acid sequence=SEQ ID NO: 372 Amino acid sequence=SEQID NO: 1072; Antibody Heavy Chain No:192622 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 373 Amino acid sequence=SEQ ID NO: 1073; Antibody Heavy ChainNo:4423 germ=IGHV1-2*02 seqid1=47.9 seqid3=47.7 thrd1=0.160 thrd3=0.155divg=0.132 Nucleic acid sequence=SEQ ID NO: 374 Amino acid sequence=SEQID NO: 1074; Antibody Heavy Chain No:39390 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 375 Amino acid sequence=SEQ ID NO: 1075; Antibody Heavy ChainNo:121691 germ=IGHV1-2*02 seqid1=45.5 seqid3=47.7 thrd1=0.181thrd3=0.183 divg=0.132 Nucleic acid sequence=SEQ ID NO: 376 Amino acidsequence=SEQ ID NO: 1076; Antibody Heavy Chain No:114705 germ=IGHV1-2*02seqid1=47.9 seqid3=47.7 thrd1=0.162 thrd3=0.157 divg=0.132 Nucleic acidsequence=SEQ ID NO: 377 Amino acid sequence=SEQ ID NO: 1077; AntibodyHeavy Chain No:66658 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 378 Amino acidsequence=SEQ ID NO: 1078; Antibody Heavy Chain No:20622 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 379 Amino acid sequence=SEQ ID NO: 1079; AntibodyHeavy Chain No:7790 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 380 Amino acidsequence=SEQ ID NO: 1080; Antibody Heavy Chain No:74326 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 381 Amino acid sequence=SEQ ID NO: 1081; AntibodyHeavy Chain No:62399 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 382 Amino acidsequence=SEQ ID NO: 1082; Antibody Heavy Chain No:90523 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 383 Amino acid sequence=SEQ ID NO: 1083; AntibodyHeavy Chain No:86744 germ=IGHV1-2*02 seqid1=52.1 seqid3=46.2 thrd1=0.101thrd3=0.140 divg=0.132 Nucleic acid sequence=SEQ ID NO: 384 Amino acidsequence=SEQ ID NO: 1084; Antibody Heavy Chain No:65521 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 385 Amino acid sequence=SEQ ID NO: 1085; AntibodyHeavy Chain No:25587 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 386 Amino acidsequence=SEQ ID NO: 1086; Antibody Heavy Chain No:93763 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 387 Amino acid sequence=SEQ ID NO: 1087; AntibodyHeavy Chain No:8204 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 388 Amino acidsequence=SEQ ID NO: 1088; Antibody Heavy Chain No:32439 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acidsequence=SEQ ID NO: 389 Amino acid sequence=SEQ ID NO: 1089; AntibodyHeavy Chain No:29882 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 390 Amino acidsequence=SEQ ID NO: 1090; Antibody Heavy Chain No:33693 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 391 Amino acid sequence=SEQ ID NO: 1091; AntibodyHeavy Chain No:13076 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 392 Amino acidsequence=SEQ ID NO: 1092; Antibody Heavy Chain No:31565 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acidsequence=SEQ ID NO: 393 Amino acid sequence=SEQ ID NO: 1093; AntibodyHeavy Chain No:114518 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQ ID NO: 394Amino acid sequence=SEQ ID NO: 1094; Antibody Heavy Chain No:55150germ=IGHV1-2*02 seqid1=39.7 seqid3=40.8 thrd1=0.204 thrd3=0.313divg=0.132 Nucleic acid sequence=SEQ ID NO: 395 Amino acid sequence=SEQID NO: 1095; Antibody Heavy Chain No:83555 germ=IGHV1-2*02 seqid1=47.9seqid3=47.7 thrd1=0.162 thrd3=0.157 divg=0.132 Nucleic acid sequence=SEQID NO: 396 Amino acid sequence=SEQ ID NO: 1096; Antibody Heavy ChainNo:57699 germ=IGHV1-2*02 seqid1=52.9 seqid3=46.9 thrd1=0.192 thrd3=0.214divg=0.132 Nucleic acid sequence=SEQ ID NO: 397 Amino acid sequence=SEQID NO: 1097; Antibody Heavy Chain No:46426 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 398 Amino acid sequence=SEQ ID NO: 1098; Antibody Heavy ChainNo:50130 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 399 Amino acid sequence=SEQID NO: 1099; Antibody Heavy Chain No:17965 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 400 Amino acid sequence=SEQ ID NO: 1100; Antibody Heavy ChainNo:40699 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 401 Amino acid sequence=SEQID NO: 1101; Antibody Heavy Chain No:17552 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 402 Amino acid sequence=SEQ ID NO: 1102; Antibody Heavy ChainNo:73425 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 403 Amino acid sequence=SEQID NO: 1103; Antibody Heavy Chain No:20437 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQID NO: 404 Amino acid sequence=SEQ ID NO: 1104; Antibody Heavy ChainNo:59091 germ=IGHV1-2*02 seqid1=49.6 seqid3=49.2 thrd1=0.158 thrd3=0.154divg=0.132 Nucleic acid sequence=SEQ ID NO: 405 Amino acid sequence=SEQID NO: 1105; Antibody Heavy Chain No:21770 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.132 Nucleic acid sequence=SEQID NO: 406 Amino acid sequence=SEQ ID NO: 1106; Antibody Heavy ChainNo:11760 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 407 Amino acid sequence=SEQID NO: 1107; Antibody Heavy Chain No:92232 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 408 Amino acid sequence=SEQ ID NO: 1108; Antibody Heavy ChainNo:77905 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.132 Nucleic acid sequence=SEQ ID NO: 409 Amino acid sequence=SEQID NO: 1109; Antibody Heavy Chain No:114286 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 410 Amino acid sequence=SEQ ID NO: 1110; Antibody Heavy ChainNo:17493 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.132 Nucleic acid sequence=SEQ ID NO: 411 Amino acid sequence=SEQID NO: 1111; Antibody Heavy Chain No:134356 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.132 Nucleic acid sequence=SEQID NO: 412 Amino acid sequence=SEQ ID NO: 1112; Antibody Heavy ChainNo:55840 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 413 Amino acid sequence=SEQID NO: 1113; Antibody Heavy Chain No:20575 germ=IGHV1-2*02 seqid1=43.8seqid3=45.4 thrd1=0.253 thrd3=0.185 divg=0.128 Nucleic acid sequence=SEQID NO: 414 Amino acid sequence=SEQ ID NO: 1114; Antibody Heavy ChainNo:31418 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 415 Amino acid sequence=SEQID NO: 1115; Antibody Heavy Chain No:25111 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 416 Amino acid sequence=SEQ ID NO: 1116; Antibody Heavy ChainNo:6508 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 417 Amino acid sequence=SEQID NO: 1117; Antibody Heavy Chain No:34999 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 418 Amino acid sequence=SEQ ID NO: 1118; Antibody Heavy ChainNo:46977 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 419 Amino acid sequence=SEQID NO: 1119; Antibody Heavy Chain No:16971 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.128 Nucleic acid sequence=SEQID NO: 420 Amino acid sequence=SEQ ID NO: 1120; Antibody Heavy ChainNo:42695 germ=IGHV1-2*02 seqid1=52.1 seqid3=47.7 thrd1=0.187 thrd3=0.130divg=0.128 Nucleic acid sequence=SEQ ID NO: 421 Amino acid sequence=SEQID NO: 1121; Antibody Heavy Chain No:40345 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 422 Amino acid sequence=SEQ ID NO: 1122; Antibody Heavy ChainNo:12622 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 423 Amino acid sequence=SEQID NO: 1123; Antibody Heavy Chain No:17063 germ=IGHV1-2*02 seqid1=51.2seqid3=50.8 thrd1=0.172 thrd3=0.145 divg=0.128 Nucleic acid sequence=SEQID NO: 424 Amino acid sequence=SEQ ID NO: 1124; Antibody Heavy ChainNo:49945 germ=IGHV1-2*02 seqid1=50.4 seqid3=44.6 thrd1=0.105 thrd3=0.149divg=0.128 Nucleic acid sequence=SEQ ID NO: 425 Amino acid sequence=SEQID NO: 1125; Antibody Heavy Chain No:47052 germ=IGHV1-2*02 seqid1=52.9seqid3=49.2 thrd1=0.117 thrd3=0.117 divg=0.128 Nucleic acid sequence=SEQID NO: 426 Amino acid sequence=SEQ ID NO: 1126; Antibody Heavy ChainNo:63586 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 427 Amino acid sequence=SEQID NO: 1127; Antibody Heavy Chain No:92372 germ=IGHV1-2*02 seqid1=52.9seqid3=50.0 thrd1=0.170 thrd3=0.157 divg=0.128 Nucleic acid sequence=SEQID NO: 428 Amino acid sequence=SEQ ID NO: 1128; Antibody Heavy ChainNo:31043 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 429 Amino acid sequence=SEQID NO: 1129; Antibody Heavy Chain No:10325 germ=IGHV1-2*02 seqid1=50.4seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.128 Nucleic acid sequence=SEQID NO: 430 Amino acid sequence=SEQ ID NO: 1130; Antibody Heavy ChainNo:34950 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 431 Amino acid sequence=SEQID NO: 1131; Antibody Heavy Chain No:31131 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 432 Amino acid sequence=SEQ ID NO: 1132; Antibody Heavy ChainNo:35353 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 433 Amino acid sequence=SEQID NO: 1133; Antibody Heavy Chain No:46948 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 434 Amino acid sequence=SEQ ID NO: 1134; Antibody Heavy ChainNo:65582 germ=IGHV1-2*02 seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156divg=0.128 Nucleic acid sequence=SEQ ID NO: 435 Amino acid sequence=SEQID NO: 1135; Antibody Heavy Chain No:79430 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 436 Amino acid sequence=SEQ ID NO: 1136; Antibody Heavy ChainNo:35657 germ=IGHV1-2*02 seqid1=50.4 seqid3=46.9 thrd1=0.162 thrd3=0.185divg=0.128 Nucleic acid sequence=SEQ ID NO: 437 Amino acid sequence=SEQID NO: 1137; Antibody Heavy Chain No:60213 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 438 Amino acid sequence=SEQ ID NO: 1138; Antibody Heavy ChainNo:114115 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQ ID NO: 439 Amino acidsequence=SEQ ID NO: 1139; Antibody Heavy Chain No:33812 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acidsequence=SEQ ID NO: 440 Amino acid sequence=SEQ ID NO: 1140; AntibodyHeavy Chain No:8579 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQ ID NO: 441 Amino acidsequence=SEQ ID NO: 1141; Antibody Heavy Chain No:139116 germ=IGHV1-2*02seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acidsequence=SEQ ID NO: 442 Amino acid sequence=SEQ ID NO: 1142; AntibodyHeavy Chain No:22596 germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQ ID NO: 443 Amino acidsequence=SEQ ID NO: 1143; Antibody Heavy Chain No:34400 germ=IGHV1-2*02seqid1=50.4 seqid3=50.0 thrd1=0.161 thrd3=0.156 divg=0.128 Nucleic acidsequence=SEQ ID NO: 444 Amino acid sequence=SEQ ID NO: 1144; AntibodyHeavy Chain No:115582 germ=IGHV1-2*02 seqid1=52.1 seqid3=47.7thrd1=0.187 thrd3=0.130 divg=0.128 Nucleic acid sequence=SEQ ID NO: 445Amino acid sequence=SEQ ID NO: 1145; Antibody Heavy Chain No:53897germ=IGHV1-2*02 seqid1=53.7 seqid3=47.7 thrd1=0.093 thrd3=0.133divg=0.128 Nucleic acid sequence=SEQ ID NO: 446 Amino acid sequence=SEQID NO: 1146; Antibody Heavy Chain No:18972 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 447 Amino acid sequence=SEQ ID NO: 1147; Antibody Heavy ChainNo:38620 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.160 thrd3=0.155divg=0.128 Nucleic acid sequence=SEQ ID NO: 448 Amino acid sequence=SEQID NO: 1148; Antibody Heavy Chain No:109187 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 449 Amino acid sequence=SEQ ID NO: 1149; Antibody Heavy ChainNo:14179 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.160 thrd3=0.155divg=0.128 Nucleic acid sequence=SEQ ID NO: 450 Amino acid sequence=SEQID NO: 1150; Antibody Heavy Chain No:66584 germ=IGHV1-2*02 seqid1=53.7seqid3=47.7 thrd1=0.093 thrd3=0.133 divg=0.128 Nucleic acid sequence=SEQID NO: 451 Amino acid sequence=SEQ ID NO: 1151; Antibody Heavy ChainNo:7295 germ=IGHV1-2*02 seqid1=49.6 seqid3=51.5 thrd1=0.154 thrd3=0.196divg=0.128 Nucleic acid sequence=SEQ ID NO: 452 Amino acid sequence=SEQID NO: 1152; Antibody Heavy Chain No:82463 germ=IGHV1-2*02 seqid1=54.5seqid3=50.8 thrd1=0.150 thrd3=0.120 divg=0.128 Nucleic acid sequence=SEQID NO: 453 Amino acid sequence=SEQ ID NO: 1153; Antibody Heavy ChainNo:142429 germ=IGHV1-2*02 seqid1=53.7 seqid3=46.2 thrd1=0.100thrd3=0.138 divg=0.128 Nucleic acid sequence=SEQ ID NO: 454 Amino acidsequence=SEQ ID NO: 1154; Antibody Heavy Chain No:39408 germ=IGHV1-2*02seqid1=44.6 seqid3=46.2 thrd1=0.218 thrd3=0.168 divg=0.125 Nucleic acidsequence=SEQ ID NO: 455 Amino acid sequence=SEQ ID NO: 1155; AntibodyHeavy Chain No:71215 germ=IGHV1-2*02 seqid1=43.8 seqid3=45.4 thrd1=0.219thrd3=0.167 divg=0.125 Nucleic acid sequence=SEQ ID NO: 456 Amino acidsequence=SEQ ID NO: 1156; Antibody Heavy Chain No:9788 germ=IGHV1-2*02seqid1=49.6 seqid3=46.2 thrd1=0.188 thrd3=0.174 divg=0.125 Nucleic acidsequence=SEQ ID NO: 457 Amino acid sequence=SEQ ID NO: 1157; AntibodyHeavy Chain No:37061 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.172thrd3=0.226 divg=0.125 Nucleic acid sequence=SEQ ID NO: 458 Amino acidsequence=SEQ ID NO: 1158; Antibody Heavy Chain No:115015 germ=IGHV1-2*02seqid1=53.7 seqid3=50.8 thrd1=0.147 thrd3=0.131 divg=0.125 Nucleic acidsequence=SEQ ID NO: 459 Amino acid sequence=SEQ ID NO: 1159; AntibodyHeavy Chain No:46793 germ=IGHV1-2*02 seqid1=44.6 seqid3=46.2 thrd1=0.247thrd3=0.168 divg=0.125 Nucleic acid sequence=SEQ ID NO: 460 Amino acidsequence=SEQ ID NO: 1160; Antibody Heavy Chain No:80516 germ=IGHV1-2*02seqid1=47.1 seqid3=45.4 thrd1=0.187 thrd3=0.204 divg=0.125 Nucleic acidsequence=SEQ ID NO: 461 Amino acid sequence=SEQ ID NO: 1161; AntibodyHeavy Chain No:76415 germ=IGHV1-2*02 seqid1=49.6 seqid3=47.7 thrd1=0.196thrd3=0.218 divg=0.125 Nucleic acid sequence=SEQ ID NO: 462 Amino acidsequence=SEQ ID NO: 1162; Antibody Heavy Chain No:154569 germ=IGHV1-2*02seqid1=47.9 seqid3=49.2 thrd1=0.206 thrd3=0.150 divg=0.125 Nucleic acidsequence=SEQ ID NO: 463 Amino acid sequence=SEQ ID NO: 1163; AntibodyHeavy Chain No:104939 germ=IGHV1-2*02 seqid1=52.1 seqid3=53.8thrd1=0.176 thrd3=0.128 divg=0.122 Nucleic acid sequence=SEQ ID NO: 464Amino acid sequence=SEQ ID NO: 1164; Antibody Heavy Chain No:92580germ=IGHV1-2*02 seqid1=49.6 seqid3=48.5 thrd1=0.190 thrd3=0.147divg=0.122 Nucleic acid sequence=SEQ ID NO: 465 Amino acid sequence=SEQID NO: 1165; Antibody Heavy Chain No:30625 germ=IGHV1-2*02 seqid1=53.7seqid3=50.0 thrd1=0.146 thrd3=0.132 divg=0.122 Nucleic acid sequence=SEQID NO: 466 Amino acid sequence=SEQ ID NO: 1166; Antibody Heavy ChainNo:91867 germ=IGHV1-2*02 seqid1=51.2 seqid3=47.7 thrd1=0.225 thrd3=0.156divg=0.122 Nucleic acid sequence=SEQ ID NO: 467 Amino acid sequence=SEQID NO: 1167; Antibody Heavy Chain No:62307 germ=IGHV1-2*02 seqid1=48.8seqid3=47.7 thrd1=0.172 thrd3=0.164 divg=0.122 Nucleic acid sequence=SEQID NO: 468 Amino acid sequence=SEQ ID NO: 1168; Antibody Heavy ChainNo:26484 germ=IGHV1-2*02 seqid1=54.5 seqid3=47.7 thrd1=0.183 thrd3=0.154divg=0.122 Nucleic acid sequence=SEQ ID NO: 469 Amino acid sequence=SEQID NO: 1169; Antibody Heavy Chain No:40754 germ=IGHV1-2*02 seqid1=52.1seqid3=50.8 thrd1=0.163 thrd3=0.159 divg=0.122 Nucleic acid sequence=SEQID NO: 470 Amino acid sequence=SEQ ID NO: 1170; Antibody Heavy ChainNo:151057 germ=IGHV1-2*02 seqid1=50.4 seqid3=52.3 thrd1=0.152thrd3=0.191 divg=0.122 Nucleic acid sequence=SEQ ID NO: 471 Amino acidsequence=SEQ ID NO: 1171; Antibody Heavy Chain No:51285 germ=IGHV1-2*02seqid1=53.7 seqid3=44.6 thrd1=0.204 thrd3=0.155 divg=0.122 Nucleic acidsequence=SEQ ID NO: 472 Amino acid sequence=SEQ ID NO: 1172; AntibodyHeavy Chain No:23038 germ=IGHV1-2*02 seqid1=52.1 seqid3=50.0 thrd1=0.147thrd3=0.135 divg=0.122 Nucleic acid sequence=SEQ ID NO: 473 Amino acidsequence=SEQ ID NO: 1173; Antibody Heavy Chain No:118516 germ=IGHV1-2*02seqid1=53.7 seqid3=50.8 thrd1=0.158 thrd3=0.114 divg=0.122 Nucleic acidsequence=SEQ ID NO: 474 Amino acid sequence=SEQ ID NO: 1174; AntibodyHeavy Chain No:54561 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.173thrd3=0.165 divg=0.122 Nucleic acid sequence=SEQ ID NO: 475 Amino acidsequence=SEQ ID NO: 1175; Antibody Heavy Chain No:7907 germ=IGHV1-2*02seqid1=47.1 seqid3=48.5 thrd1=0.216 thrd3=0.163 divg=0.122 Nucleic acidsequence=SEQ ID NO: 476 Amino acid sequence=SEQ ID NO: 1176; AntibodyHeavy Chain No:48955 germ=IGHV1-2*02 seqid1=52.9 seqid3=44.6 thrd1=0.208thrd3=0.161 divg=0.122 Nucleic acid sequence=SEQ ID NO: 477 Amino acidsequence=SEQ ID NO: 1177; Antibody Heavy Chain No:124460 germ=IGHV1-2*02seqid1=45.5 seqid3=46.9 thrd1=0.216 thrd3=0.165 divg=0.118 Nucleic acidsequence=SEQ ID NO: 478 Amino acid sequence=SEQ ID NO: 1178; AntibodyHeavy Chain No:60838 germ=IGHV1-2*02 seqid1=45.5 seqid3=45.4 thrd1=0.188thrd3=0.145 divg=0.118 Nucleic acid sequence=SEQ ID NO: 479 Amino acidsequence=SEQ ID NO: 1179; Antibody Heavy Chain No:9327 germ=IGHV1-2*02seqid1=54.5 seqid3=50.8 thrd1=0.136 thrd3=0.112 divg=0.118 Nucleic acidsequence=SEQ ID NO: 480 Amino acid sequence=SEQ ID NO: 1180; AntibodyHeavy Chain No:12603 germ=IGHV1-2*02 seqid1=44.6 seqid3=46.2 thrd1=0.218thrd3=0.168 divg=0.118 Nucleic acid sequence=SEQ ID NO: 481 Amino acidsequence=SEQ ID NO: 1181; Antibody Heavy Chain No:67294 germ=IGHV1-2*02seqid1=41.3 seqid3=43.8 thrd1=0.199 thrd3=0.390 divg=0.118 Nucleic acidsequence=SEQ ID NO: 482 Amino acid sequence=SEQ ID NO: 1182; AntibodyHeavy Chain No:31882 germ=IGHV1-2*02 seqid1=43.8 seqid3=45.4 thrd1=0.212thrd3=0.160 divg=0.118 Nucleic acid sequence=SEQ ID NO: 483 Amino acidsequence=SEQ ID NO: 1183; Antibody Heavy Chain No:7187 germ=IGHV1-2*02seqid1=47.1 seqid3=48.5 thrd1=0.216 thrd3=0.163 divg=0.118 Nucleic acidsequence=SEQ ID NO: 484 Amino acid sequence=SEQ ID NO: 1184; AntibodyHeavy Chain No:11340 germ=IGHV1-2*02 seqid1=48.8 seqid3=47.7 thrd1=0.150thrd3=0.118 divg=0.118 Nucleic acid sequence=SEQ ID NO: 485 Amino acidsequence=SEQ ID NO: 1185; Antibody Heavy Chain No:21889 germ=IGHV1-2*02seqid1=54.5 seqid3=50.0 thrd1=0.153 thrd3=0.108 divg=0.118 Nucleic acidsequence=SEQ ID NO: 486 Amino acid sequence=SEQ ID NO: 1186; AntibodyHeavy Chain No:48803 germ=IGHV1-2*02 seqid1=53.7 seqid3=50.8 thrd1=0.158thrd3=0.114 divg=0.118 Nucleic acid sequence=SEQ ID NO: 487 Amino acidsequence=SEQ ID NO: 1187; Antibody Heavy Chain No:26563 germ=IGHV1-2*02seqid1=43.8 seqid3=45.4 thrd1=0.212 thrd3=0.160 divg=0.118 Nucleic acidsequence=SEQ ID NO: 488 Amino acid sequence=SEQ ID NO: 1188; AntibodyHeavy Chain No:5049 germ=IGHV1-2*02 seqid1=54.5 seqid3=50.8 thrd1=0.153thrd3=0.111 divg=0.118 Nucleic acid sequence=SEQ ID NO: 489 Amino acidsequence=SEQ ID NO: 1189; Antibody Heavy Chain No:52934 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.207 thrd3=0.173 divg=0.118 Nucleic acidsequence=SEQ ID NO: 490 Amino acid sequence=SEQ ID NO: 1190; AntibodyHeavy Chain No:131623 germ=IGHV1-2*02 seqid1=46.3 seqid3=48.5thrd1=0.217 thrd3=0.163 divg=0.118 Nucleic acid sequence=SEQ ID NO: 491Amino acid sequence=SEQ ID NO: 1191; Antibody Heavy Chain No:32431germ=IGHV1-2*02 seqid1=48.8 seqid3=44.6 thrd1=0.198 thrd3=0.144divg=0.118 Nucleic acid sequence=SEQ ID NO: 492 Amino acid sequence=SEQID NO: 1192; Antibody Heavy Chain No:49222 germ=IGHV1-2*02 seqid1=52.9seqid3=49.2 thrd1=0.156 thrd3=0.168 divg=0.118 Nucleic acid sequence=SEQID NO: 493 Amino acid sequence=SEQ ID NO: 1193; Antibody Heavy ChainNo:7766 germ=IGHV1-2*02 seqid1=47.1 seqid3=48.5 thrd1=0.214 thrd3=0.161divg=0.118 Nucleic acid sequence=SEQ ID NO: 494 Amino acid sequence=SEQID NO: 1194; Antibody Heavy Chain No:111944 germ=IGHV1-2*02 seqid1=44.6seqid3=46.2 thrd1=0.247 thrd3=0.168 divg=0.118 Nucleic acid sequence=SEQID NO: 495 Amino acid sequence=SEQ ID NO: 1195; Antibody Heavy ChainNo:42112 germ=IGHV1-2*02 seqid1=47.9 seqid3=49.2 thrd1=0.167 thrd3=0.208divg=0.118 Nucleic acid sequence=SEQ ID NO: 496 Amino acid sequence=SEQID NO: 1196; Antibody Heavy Chain No:130294 germ=IGHV1-2*02 seqid1=49.6seqid3=48.5 thrd1=0.149 thrd3=0.117 divg=0.115 Nucleic acid sequence=SEQID NO: 497 Amino acid sequence=SEQ ID NO: 1197; Antibody Heavy ChainNo:8818 germ=IGHV1-2*02 seqid1=48.8 seqid3=45.4 thrd1=0.193 thrd3=0.143divg=0.115 Nucleic acid sequence=SEQ ID NO: 498 Amino acid sequence=SEQID NO: 1198; Antibody Heavy Chain No:84071 germ=IGHV1-2*02 seqid1=49.6seqid3=50.8 thrd1=0.165 thrd3=0.215 divg=0.115 Nucleic acid sequence=SEQID NO: 499 Amino acid sequence=SEQ ID NO: 1199; Antibody Heavy ChainNo:25068 germ=IGHV1-2*02 seqid1=53.7 seqid3=50.8 thrd1=0.158 thrd3=0.114divg=0.115 Nucleic acid sequence=SEQ ID NO: 500 Amino acid sequence=SEQID NO: 1200; Antibody Heavy Chain No:2768 germ=IGHV1-2*02 seqid1=46.3seqid3=43.1 thrd1=0.187 thrd3=0.305 divg=0.115 Nucleic acid sequence=SEQID NO: 501 Amino acid sequence=SEQ ID NO: 1201; Antibody Heavy ChainNo:16010 germ=IGHV1-2*02 seqid1=52.9 seqid3=50.0 thrd1=0.150 thrd3=0.166divg=0.115 Nucleic acid sequence=SEQ ID NO: 502 Amino acid sequence=SEQID NO: 1202; Antibody Heavy Chain No:60573 germ=IGHV1-2*02 seqid1=48.8seqid3=48.5 thrd1=0.181 thrd3=0.151 divg=0.115 Nucleic acid sequence=SEQID NO: 503 Amino acid sequence=SEQ ID NO: 1203; Antibody Heavy ChainNo:75010 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.8 thrd1=0.165 thrd3=0.215divg=0.115 Nucleic acid sequence=SEQ ID NO: 504 Amino acid sequence=SEQID NO: 1204; Antibody Heavy Chain No:56346 germ=IGHV1-2*02 seqid1=52.9seqid3=50.0 thrd1=0.150 thrd3=0.166 divg=0.115 Nucleic acid sequence=SEQID NO: 505 Amino acid sequence=SEQ ID NO: 1205; Antibody Heavy ChainNo:15961 germ=IGHV1-2*02 seqid1=49.6 seqid3=51.5 thrd1=0.164 thrd3=0.202divg=0.115 Nucleic acid sequence=SEQ ID NO: 506 Amino acid sequence=SEQID NO: 1206; Antibody Heavy Chain No:6826 germ=IGHV1-2*02 seqid1=51.2seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.115 Nucleic acid sequence=SEQID NO: 507 Amino acid sequence=SEQ ID NO: 1207; Antibody Heavy ChainNo:64926 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.8 thrd1=0.165 thrd3=0.215divg=0.115 Nucleic acid sequence=SEQ ID NO: 508 Amino acid sequence=SEQID NO: 1208; Antibody Heavy Chain No:79151 germ=IGHV1-2*02 seqid1=46.3seqid3=46.2 thrd1=0.183 thrd3=0.146 divg=0.115 Nucleic acid sequence=SEQID NO: 509 Amino acid sequence=SEQ ID NO: 1209; Antibody Heavy ChainNo:30800 germ=IGHV1-2*02 seqid1=53.7 seqid3=50.0 thrd1=0.155 thrd3=0.108divg=0.115 Nucleic acid sequence=SEQ ID NO: 510 Amino acid sequence=SEQID NO: 1210; Antibody Heavy Chain No:77050 germ=IGHV1-2*02 seqid1=50.4seqid3=49.2 thrd1=0.175 thrd3=0.188 divg=0.115 Nucleic acid sequence=SEQID NO: 511 Amino acid sequence=SEQ ID NO: 1211; Antibody Heavy ChainNo:26431 germ=IGHV1-2*02 seqid1=46.3 seqid3=46.2 thrd1=0.187 thrd3=0.143divg=0.115 Nucleic acid sequence=SEQ ID NO: 512 Amino acid sequence=SEQID NO: 1212; Antibody Heavy Chain No:138734 germ=IGHV1-2*02 seqid1=45.5seqid3=37.7 thrd1=0.220 thrd3=0.247 divg=0.115 Nucleic acid sequence=SEQID NO: 513 Amino acid sequence=SEQ ID NO: 1213; Antibody Heavy ChainNo:25677 germ=IGHV1-2*02 seqid1=50.4 seqid3=48.5 thrd1=0.122 thrd3=0.137divg=0.115 Nucleic acid sequence=SEQ ID NO: 514 Amino acid sequence=SEQID NO: 1214; Antibody Heavy Chain No:11877 germ=IGHV1-2*02 seqid1=53.7seqid3=50.8 thrd1=0.158 thrd3=0.114 divg=0.115 Nucleic acid sequence=SEQID NO: 515 Amino acid sequence=SEQ ID NO: 1215; Antibody Heavy ChainNo:24914 germ=IGHV1-2*02 seqid1=48.8 seqid3=48.5 thrd1=0.191 thrd3=0.163divg=0.111 Nucleic acid sequence=SEQ ID NO: 516 Amino acid sequence=SEQID NO: 1216; Antibody Heavy Chain No:66930 germ=IGHV1-2*02 seqid1=44.6seqid3=43.8 thrd1=0.218 thrd3=0.191 divg=0.111 Nucleic acid sequence=SEQID NO: 517 Amino acid sequence=SEQ ID NO: 1217; Antibody Heavy ChainNo:120736 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.158thrd3=0.206 divg=0.111 Nucleic acid sequence=SEQ ID NO: 518 Amino acidsequence=SEQ ID NO: 1218; Antibody Heavy Chain No:74892 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.184 thrd3=0.161 divg=0.111 Nucleic acidsequence=SEQ ID NO: 519 Amino acid sequence=SEQ ID NO: 1219; AntibodyHeavy Chain No:62366 germ=IGHV1-2*02 seqid1=52.1 seqid3=50.0 thrd1=0.183thrd3=0.196 divg=0.111 Nucleic acid sequence=SEQ ID NO: 520 Amino acidsequence=SEQ ID NO: 1220; Antibody Heavy Chain No:2779 germ=IGHV1-2*02seqid1=51.2 seqid3=49.2 thrd1=0.176 thrd3=0.168 divg=0.111 Nucleic acidsequence=SEQ ID NO: 521 Amino acid sequence=SEQ ID NO: 1221; AntibodyHeavy Chain No:79804 germ=IGHV1-2*02 seqid1=50.4 seqid3=51.5 thrd1=0.197thrd3=0.167 divg=0.111 Nucleic acid sequence=SEQ ID NO: 522 Amino acidsequence=SEQ ID NO: 1222; Antibody Heavy Chain No:26006 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.184 thrd3=0.161 divg=0.111 Nucleic acidsequence=SEQ ID NO: 523 Amino acid sequence=SEQ ID NO: 1223; AntibodyHeavy Chain No:35261 germ=IGHV1-2*02 seqid1=52.1 seqid3=46.9 thrd1=0.156thrd3=0.204 divg=0.111 Nucleic acid sequence=SEQ ID NO: 524 Amino acidsequence=SEQ ID NO: 1224; Antibody Heavy Chain No:32667 germ=IGHV1-2*02seqid1=47.1 seqid3=47.7 thrd1=0.218 thrd3=0.142 divg=0.108 Nucleic acidsequence=SEQ ID NO: 525 Amino acid sequence=SEQ ID NO: 1225; AntibodyHeavy Chain No:100962 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 526Amino acid sequence=SEQ ID NO: 1226; Antibody Heavy Chain No:162078germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160divg=0.108 Nucleic acid sequence=SEQ ID NO: 527 Amino acid sequence=SEQID NO: 1227; Antibody Heavy Chain No:6353 germ=IGHV1-2*02 seqid1=51.2seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQID NO: 528 Amino acid sequence=SEQ ID NO: 1228; Antibody Heavy ChainNo:52276 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160divg=0.108 Nucleic acid sequence=SEQ ID NO: 529 Amino acid sequence=SEQID NO: 1229; Antibody Heavy Chain No:60197 germ=IGHV1-2*02 seqid1=47.1seqid3=43.8 thrd1=0.149 thrd3=0.226 divg=0.108 Nucleic acid sequence=SEQID NO: 530 Amino acid sequence=SEQ ID NO: 1230; Antibody Heavy ChainNo:59165 germ=IGHV1-2*02 seqid1=47.9 seqid3=49.2 thrd1=0.186 thrd3=0.164divg=0.108 Nucleic acid sequence=SEQ ID NO: 531 Amino acid sequence=SEQID NO: 1231; Antibody Heavy Chain No:69714 germ=IGHV1-2*02 seqid1=53.7seqid3=52.3 thrd1=0.156 thrd3=0.146 divg=0.108 Nucleic acid sequence=SEQID NO: 532 Amino acid sequence=SEQ ID NO: 1232; Antibody Heavy ChainNo:19770 germ=IGHV1-2*02 seqid1=52.1 seqid3=51.5 thrd1=0.184 thrd3=0.159divg=0.108 Nucleic acid sequence=SEQ ID NO: 533 Amino acid sequence=SEQID NO: 1233; Antibody Heavy Chain No:72439 germ=IGHV1-2*02 seqid1=47.9seqid3=49.2 thrd1=0.184 thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQID NO: 534 Amino acid sequence=SEQ ID NO: 1234; Antibody Heavy ChainNo:22551 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.0 thrd1=0.207 thrd3=0.177divg=0.108 Nucleic acid sequence=SEQ ID NO: 535 Amino acid sequence=SEQID NO: 1235; Antibody Heavy Chain No:61263 germ=IGHV1-2*02 seqid1=51.2seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQID NO: 536 Amino acid sequence=SEQ ID NO: 1236; Antibody Heavy ChainNo:40709 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160divg=0.108 Nucleic acid sequence=SEQ ID NO: 537 Amino acid sequence=SEQID NO: 1237; Antibody Heavy Chain No:20145 germ=IGHV1-2*02 seqid1=52.1seqid3=51.5 thrd1=0.184 thrd3=0.159 divg=0.108 Nucleic acid sequence=SEQID NO: 538 Amino acid sequence=SEQ ID NO: 1238; Antibody Heavy ChainNo:150344 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.158thrd3=0.206 divg=0.108 Nucleic acid sequence=SEQ ID NO: 539 Amino acidsequence=SEQ ID NO: 1239; Antibody Heavy Chain No:107592 germ=IGHV1-2*02seqid1=52.1 seqid3=45.4 thrd1=0.123 thrd3=0.157 divg=0.108 Nucleic acidsequence=SEQ ID NO: 540 Amino acid sequence=SEQ ID NO: 1240; AntibodyHeavy Chain No:35068 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 541 Amino acidsequence=SEQ ID NO: 1241; Antibody Heavy Chain No:34274 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acidsequence=SEQ ID NO: 542 Amino acid sequence=SEQ ID NO: 1242; AntibodyHeavy Chain No:44773 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 543 Amino acidsequence=SEQ ID NO: 1243; Antibody Heavy Chain No:41319 germ=IGHV1-2*02seqid1=48.8 seqid3=48.5 thrd1=0.206 thrd3=0.183 divg=0.108 Nucleic acidsequence=SEQ ID NO: 544 Amino acid sequence=SEQ ID NO: 1244; AntibodyHeavy Chain No:42823 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.158thrd3=0.206 divg=0.108 Nucleic acid sequence=SEQ ID NO: 545 Amino acidsequence=SEQ ID NO: 1245; Antibody Heavy Chain No:41670 germ=IGHV1-2*02seqid1=55.4 seqid3=47.7 thrd1=0.124 thrd3=0.148 divg=0.108 Nucleic acidsequence=SEQ ID NO: 546 Amino acid sequence=SEQ ID NO: 1246; AntibodyHeavy Chain No:45717 germ=IGHV1-2*02 seqid1=49.6 seqid3=46.9 thrd1=0.155thrd3=0.206 divg=0.108 Nucleic acid sequence=SEQ ID NO: 547 Amino acidsequence=SEQ ID NO: 1247; Antibody Heavy Chain No:26052 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acidsequence=SEQ ID NO: 548 Amino acid sequence=SEQ ID NO: 1248; AntibodyHeavy Chain No:96832 germ=IGHV1-2*02 seqid1=47.9 seqid3=45.4 thrd1=0.173thrd3=0.258 divg=0.108 Nucleic acid sequence=SEQ ID NO: 549 Amino acidsequence=SEQ ID NO: 1249; Antibody Heavy Chain No:118499 germ=IGHV1-2*02seqid1=49.6 seqid3=50.8 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acidsequence=SEQ ID NO: 550 Amino acid sequence=SEQ ID NO: 1250; AntibodyHeavy Chain No:38820 germ=IGHV1-2*02 seqid1=47.9 seqid3=49.2 thrd1=0.186thrd3=0.164 divg=0.108 Nucleic acid sequence=SEQ ID NO: 551 Amino acidsequence=SEQ ID NO: 1251; Antibody Heavy Chain No:50301 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acidsequence=SEQ ID NO: 552 Amino acid sequence=SEQ ID NO: 1252; AntibodyHeavy Chain No:18973 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 553 Amino acidsequence=SEQ ID NO: 1253; Antibody Heavy Chain No:70392 germ=IGHV1-2*02seqid1=52.1 seqid3=51.5 thrd1=0.184 thrd3=0.159 divg=0.108 Nucleic acidsequence=SEQ ID NO: 554 Amino acid sequence=SEQ ID NO: 1254; AntibodyHeavy Chain No:85662 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3 thrd1=0.185thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 555 Amino acidsequence=SEQ ID NO: 1255; Antibody Heavy Chain No:145143 germ=IGHV1-2*02seqid1=51.2 seqid3=52.3 thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acidsequence=SEQ ID NO: 556 Amino acid sequence=SEQ ID NO: 1256; AntibodyHeavy Chain No:138251 germ=IGHV1-2*02 seqid1=48.8 seqid3=46.9thrd1=0.184 thrd3=0.237 divg=0.108 Nucleic acid sequence=SEQ ID NO: 557Amino acid sequence=SEQ ID NO: 1257; Antibody Heavy Chain No:31587germ=IGHV1-2*02 seqid1=51.2 seqid3=53.1 thrd1=0.297 thrd3=0.155divg=0.108 Nucleic acid sequence=SEQ ID NO: 558 Amino acid sequence=SEQID NO: 1258; Antibody Heavy Chain No:62466 germ=IGHV1-2*02 seqid1=49.6seqid3=48.5 thrd1=0.156 thrd3=0.218 divg=0.108 Nucleic acid sequence=SEQID NO: 559 Amino acid sequence=SEQ ID NO: 1259; Antibody Heavy ChainNo:131429 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.158thrd3=0.206 divg=0.108 Nucleic acid sequence=SEQ ID NO: 560 Amino acidsequence=SEQ ID NO: 1260; Antibody Heavy Chain No:44658 germ=IGHV1-2*02seqid1=51.2 seqid3=47.7 thrd1=0.156 thrd3=0.207 divg=0.108 Nucleic acidsequence=SEQ ID NO: 561 Amino acid sequence=SEQ ID NO: 1261; AntibodyHeavy Chain No:144844 germ=IGHV1-2*02 seqid1=51.2 seqid3=52.3thrd1=0.185 thrd3=0.160 divg=0.108 Nucleic acid sequence=SEQ ID NO: 562Amino acid sequence=SEQ ID NO: 1262; Antibody Heavy Chain No:44516germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.158 thrd3=0.206divg=0.108 Nucleic acid sequence=SEQ ID NO: 563 Amino acid sequence=SEQID NO: 1263; Antibody Heavy Chain No:54766 germ=IGHV1-2*02 seqid1=52.1seqid3=46.9 thrd1=0.199 thrd3=0.152 divg=0.105 Nucleic acid sequence=SEQID NO: 564 Amino acid sequence=SEQ ID NO: 1264; Antibody Heavy ChainNo:76743 germ=IGHV1-2*02 seqid1=53.7 seqid3=49.2 thrd1=0.172 thrd3=0.194divg=0.105 Nucleic acid sequence=SEQ ID NO: 565 Amino acid sequence=SEQID NO: 1265; Antibody Heavy Chain No:104654 germ=IGHV1-2*02 seqid1=47.9seqid3=43.8 thrd1=0.169 thrd3=0.140 divg=0.105 Nucleic acid sequence=SEQID NO: 566 Amino acid sequence=SEQ ID NO: 1266; Antibody Heavy ChainNo:124687 germ=IGHV1-2*02 seqid1=47.1 seqid3=48.5 thrd1=0.189thrd3=0.166 divg=0.105 Nucleic acid sequence=SEQ ID NO: 567 Amino acidsequence=SEQ ID NO: 1267; Antibody Heavy Chain No:49649 germ=IGHV1-2*02seqid1=53.7 seqid3=49.2 thrd1=0.171 thrd3=0.193 divg=0.105 Nucleic acidsequence=SEQ ID NO: 568 Amino acid sequence=SEQ ID NO: 1268; AntibodyHeavy Chain No:74653 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2 thrd1=0.156thrd3=0.148 divg=0.105 Nucleic acid sequence=SEQ ID NO: 569 Amino acidsequence=SEQ ID NO: 1269; Antibody Heavy Chain No:106878 germ=IGHV1-2*02seqid1=49.6 seqid3=50.0 thrd1=0.186 thrd3=0.121 divg=0.105 Nucleic acidsequence=SEQ ID NO: 570 Amino acid sequence=SEQ ID NO: 1270; AntibodyHeavy Chain No:86161 germ=IGHV1-2*02 seqid1=52.1 seqid3=54.6 thrd1=0.159thrd3=0.133 divg=0.105 Nucleic acid sequence=SEQ ID NO: 571 Amino acidsequence=SEQ ID NO: 1271; Antibody Heavy Chain No:35923 germ=IGHV1-2*02seqid1=51.2 seqid3=50.8 thrd1=0.173 thrd3=0.165 divg=0.105 Nucleic acidsequence=SEQ ID NO: 572 Amino acid sequence=SEQ ID NO: 1272; AntibodyHeavy Chain No:76480 germ=IGHV1-2*02 seqid1=32.2 seqid3=36.2 thrd1=0.272thrd3=0.301 divg=0.105 Nucleic acid sequence=SEQ ID NO: 573 Amino acidsequence=SEQ ID NO: 1273; Antibody Heavy Chain No:19179 germ=IGHV1-2*02seqid1=52.1 seqid3=54.6 thrd1=0.159 thrd3=0.133 divg=0.105 Nucleic acidsequence=SEQ ID NO: 574 Amino acid sequence=SEQ ID NO: 1274; AntibodyHeavy Chain No:42178 germ=IGHV1-2*02 seqid1=52.9 seqid3=50.8 thrd1=0.312thrd3=0.189 divg=0.105 Nucleic acid sequence=SEQ ID NO: 575 Amino acidsequence=SEQ ID NO: 1275; Antibody Heavy Chain No:149342 germ=IGHV1-2*02seqid1=52.1 seqid3=48.5 thrd1=0.170 thrd3=0.140 divg=0.101 Nucleic acidsequence=SEQ ID NO: 576 Amino acid sequence=SEQ ID NO: 1276; AntibodyHeavy Chain No:12940 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.176thrd3=0.131 divg=0.101 Nucleic acid sequence=SEQ ID NO: 577 Amino acidsequence=SEQ ID NO: 1277; Antibody Heavy Chain No:50102 germ=IGHV1-2*02seqid1=54.5 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.101 Nucleic acidsequence=SEQ ID NO: 578 Amino acid sequence=SEQ ID NO: 1278; AntibodyHeavy Chain No:57948 germ=IGHV1-2*02 seqid1=52.9 seqid3=54.6 thrd1=0.159thrd3=0.136 divg=0.101 Nucleic acid sequence=SEQ ID NO: 579 Amino acidsequence=SEQ ID NO: 1279; Antibody Heavy Chain No:58689 germ=IGHV1-2*02seqid1=55.4 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.101 Nucleic acidsequence=SEQ ID NO: 580 Amino acid sequence=SEQ ID NO: 1280; AntibodyHeavy Chain No:67595 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2 thrd1=0.156thrd3=0.148 divg=0.101 Nucleic acid sequence=SEQ ID NO: 581 Amino acidsequence=SEQ ID NO: 1281; Antibody Heavy Chain No:39805 germ=IGHV1-2*02seqid1=54.5 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.101 Nucleic acidsequence=SEQ ID NO: 582 Amino acid sequence=SEQ ID NO: 1282; AntibodyHeavy Chain No:8184 germ=IGHV1-2*02 seqid1=52.1 seqid3=49.2 thrd1=0.143thrd3=0.104 divg=0.101 Nucleic acid sequence=SEQ ID NO: 583 Amino acidsequence=SEQ ID NO: 1283; Antibody Heavy Chain No:28484 germ=IGHV1-2*02seqid1=46.3 seqid3=46.2 thrd1=0.173 thrd3=0.142 divg=0.101 Nucleic acidsequence=SEQ ID NO: 584 Amino acid sequence=SEQ ID NO: 1284; AntibodyHeavy Chain No:94185 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.176thrd3=0.131 divg=0.101 Nucleic acid sequence=SEQ ID NO: 585 Amino acidsequence=SEQ ID NO: 1285; Antibody Heavy Chain No:43787 germ=IGHV1-2*02seqid1=52.1 seqid3=49.2 thrd1=0.174 thrd3=0.133 divg=0.101 Nucleic acidsequence=SEQ ID NO: 586 Amino acid sequence=SEQ ID NO: 1286; AntibodyHeavy Chain No:83620 germ=IGHV1-2*02 seqid1=55.4 seqid3=53.8 thrd1=0.243thrd3=0.147 divg=0.101 Nucleic acid sequence=SEQ ID NO: 587 Amino acidsequence=SEQ ID NO: 1287; Antibody Heavy Chain No:39478 germ=IGHV1-2*02seqid1=55.4 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.101 Nucleic acidsequence=SEQ ID NO: 588 Amino acid sequence=SEQ ID NO: 1288; AntibodyHeavy Chain No:131276 germ=IGHV1-2*02 seqid1=52.1 seqid3=48.5thrd1=0.176 thrd3=0.135 divg=0.101 Nucleic acid sequence=SEQ ID NO: 589Amino acid sequence=SEQ ID NO: 1289; Antibody Heavy Chain No:163473germ=IGHV1-2*02 seqid1=50.4 seqid3=46.2 thrd1=0.220 thrd3=0.190divg=0.101 Nucleic acid sequence=SEQ ID NO: 590 Amino acid sequence=SEQID NO: 1290; Antibody Heavy Chain No:56704 germ=IGHV1-2*02 seqid1=53.7seqid3=51.5 thrd1=0.143 thrd3=0.121 divg=0.101 Nucleic acid sequence=SEQID NO: 591 Amino acid sequence=SEQ ID NO: 1291; Antibody Heavy ChainNo:154264 germ=IGHV1-2*02 seqid1=54.5 seqid3=52.3 thrd1=0.176thrd3=0.131 divg=0.101 Nucleic acid sequence=SEQ ID NO: 592 Amino acidsequence=SEQ ID NO: 1292; Antibody Heavy Chain No:158278 germ=IGHV1-2*02seqid1=52.1 seqid3=47.7 thrd1=0.231 thrd3=0.132 divg=0.101 Nucleic acidsequence=SEQ ID NO: 593 Amino acid sequence=SEQ ID NO: 1293; AntibodyHeavy Chain No:34490 germ=IGHV1-2*02 seqid1=51.2 seqid3=49.2 thrd1=0.162thrd3=0.174 divg=0.101 Nucleic acid sequence=SEQ ID NO: 594 Amino acidsequence=SEQ ID NO: 1294; Antibody Heavy Chain No:77433 germ=IGHV1-2*02seqid1=47.9 seqid3=47.7 thrd1=0.131 thrd3=0.140 divg=0.101 Nucleic acidsequence=SEQ ID NO: 595 Amino acid sequence=SEQ ID NO: 1295; AntibodyHeavy Chain No:93604 germ=IGHV1-2*02 seqid1=51.2 seqid3=49.2 thrd1=0.159thrd3=0.203 divg=0.098 Nucleic acid sequence=SEQ ID NO: 596 Amino acidsequence=SEQ ID NO: 1296; Antibody Heavy Chain No:165480 germ=IGHV1-2*02seqid1=54.5 seqid3=50.0 thrd1=0.174 thrd3=0.195 divg=0.098 Nucleic acidsequence=SEQ ID NO: 597 Amino acid sequence=SEQ ID NO: 1297; AntibodyHeavy Chain No:89337 germ=IGHV1-2*02 seqid1=50.4 seqid3=46.2 thrd1=0.191thrd3=0.294 divg=0.098 Nucleic acid sequence=SEQ ID NO: 598 Amino acidsequence=SEQ ID NO: 1298; Antibody Heavy Chain No:141896 germ=IGHV1-2*02seqid1=51.2 seqid3=50.0 thrd1=0.179 thrd3=0.189 divg=0.098 Nucleic acidsequence=SEQ ID NO: 599 Amino acid sequence=SEQ ID NO: 1299; AntibodyHeavy Chain No:6626 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.178thrd3=0.184 divg=0.098 Nucleic acid sequence=SEQ ID NO: 600 Amino acidsequence=SEQ ID NO: 1300;Antibody Heavy Chain No:181213 germ=IGHV1-2*02 seqid1=52.1 seqid3=48.5thrd1=0.174 thrd3=0.131 divg=0.098 Nucleic acid sequence=SEQ ID NO: 601Amino acid sequence=SEQ ID NO: 1301; Antibody Heavy Chain No:22196germ=IGHV1-2*02 seqid1=54.5 seqid3=50.0 thrd1=0.174 thrd3=0.195divg=0.098 Nucleic acid sequence=SEQ ID NO: 602 Amino acid sequence=SEQID NO: 1302; Antibody Heavy Chain No:30474 germ=IGHV1-2*02 seqid1=54.5seqid3=48.5 thrd1=0.242 thrd3=0.156 divg=0.098 Nucleic acid sequence=SEQID NO: 603 Amino acid sequence=SEQ ID NO: 1303; Antibody Heavy ChainNo:125701 germ=IGHV1-2*02 seqid1=54.5 seqid3=47.7 thrd1=0.203thrd3=0.190 divg=0.098 Nucleic acid sequence=SEQ ID NO: 604 Amino acidsequence=SEQ ID NO: 1304; Antibody Heavy Chain No:9945 germ=IGHV1-2*02seqid1=56.2 seqid3=51.5 thrd1=0.160 thrd3=0.126 divg=0.098 Nucleic acidsequence=SEQ ID NO: 605 Amino acid sequence=SEQ ID NO: 1305; AntibodyHeavy Chain No:40778 germ=IGHV1-2*02 seqid1=51.2 seqid3=47.7 thrd1=0.171thrd3=0.176 divg=0.098 Nucleic acid sequence=SEQ ID NO: 606 Amino acidsequence=SEQ ID NO: 1306; Antibody Heavy Chain No:4513 germ=IGHV1-2*02seqid1=52.9 seqid3=52.3 thrd1=0.170 thrd3=0.157 divg=0.098 Nucleic acidsequence=SEQ ID NO: 607 Amino acid sequence=SEQ ID NO: 1307; AntibodyHeavy Chain No:134442 germ=IGHV1-2*02 seqid1=55.4 seqid3=53.8thrd1=0.241 thrd3=0.155 divg=0.098 Nucleic acid sequence=SEQ ID NO: 608Amino acid sequence=SEQ ID NO: 1308; Antibody Heavy Chain No:140333germ=IGHV1-2*02 seqid1=56.2 seqid3=51.5 thrd1=0.171 thrd3=0.138divg=0.098 Nucleic acid sequence=SEQ ID NO: 609 Amino acid sequence=SEQID NO: 1309; Antibody Heavy Chain No:105176 germ=IGHV1-2*02 seqid1=56.2seqid3=52.3 thrd1=0.164 thrd3=0.121 divg=0.098 Nucleic acid sequence=SEQID NO: 610 Amino acid sequence=SEQ ID NO: 1310; Antibody Heavy ChainNo:80243 germ=IGHV1-2*02 seqid1=54.5 seqid3=52.3 thrd1=0.176 thrd3=0.131divg=0.098 Nucleic acid sequence=SEQ ID NO: 611 Amino acid sequence=SEQID NO: 1311; Antibody Heavy Chain No:9911 germ=IGHV1-2*02 seqid1=53.7seqid3=52.3 thrd1=0.146 thrd3=0.136 divg=0.098 Nucleic acid sequence=SEQID NO: 612 Amino acid sequence=SEQ ID NO: 1312; Antibody Heavy ChainNo:29294 germ=IGHV1-2*02 seqid1=55.4 seqid3=47.7 thrd1=0.159 thrd3=0.120divg=0.098 Nucleic acid sequence=SEQ ID NO: 613 Amino acid sequence=SEQID NO: 1313; Antibody Heavy Chain No:34606 germ=IGHV1-2*02 seqid1=54.5seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.098 Nucleic acid sequence=SEQID NO: 614 Amino acid sequence=SEQ ID NO: 1314; Antibody Heavy ChainNo:81062 germ=IGHV1-2*02 seqid1=55.4 seqid3=47.7 thrd1=0.159 thrd3=0.120divg=0.095 Nucleic acid sequence=SEQ ID NO: 615 Amino acid sequence=SEQID NO: 1315; Antibody Heavy Chain No:57203 germ=IGHV1-2*02 seqid1=59.5seqid3=52.3 thrd1=0.134 thrd3=0.180 divg=0.095 Nucleic acid sequence=SEQID NO: 616 Amino acid sequence=SEQ ID NO: 1316; Antibody Heavy ChainNo:43280 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181 thrd3=0.193divg=0.095 Nucleic acid sequence=SEQ ID NO: 617 Amino acid sequence=SEQID NO: 1317; Antibody Heavy Chain No:115669 germ=IGHV1-2*02 seqid1=54.5seqid3=51.5 thrd1=0.183 thrd3=0.139 divg=0.095 Nucleic acid sequence=SEQID NO: 618 Amino acid sequence=SEQ ID NO: 1318; Antibody Heavy ChainNo:70426 germ=IGHV1-2*02 seqid1=53.7 seqid3=52.3 thrd1=0.144 thrd3=0.137divg=0.095 Nucleic acid sequence=SEQ ID NO: 619 Amino acid sequence=SEQID NO: 1319; Antibody Heavy Chain No:72666 germ=IGHV1-2*02 seqid1=47.9seqid3=45.4 thrd1=0.220 thrd3=0.242 divg=0.095 Nucleic acid sequence=SEQID NO: 620 Amino acid sequence=SEQ ID NO: 1320; Antibody Heavy ChainNo:119442 germ=IGHV1-2*02 seqid1=43.0 seqid3=39.2 thrd1=0.250thrd3=0.209 divg=0.095 Nucleic acid sequence=SEQ ID NO: 621 Amino acidsequence=SEQ ID NO: 1321; Antibody Heavy Chain No:143549 germ=IGHV1-2*02seqid1=51.2 seqid3=48.5 thrd1=0.222 thrd3=0.280 divg=0.095 Nucleic acidsequence=SEQ ID NO: 622 Amino acid sequence=SEQ ID NO: 1322; AntibodyHeavy Chain No:29700 germ=IGHV1-2*02 seqid1=49.6 seqid3=52.3 thrd1=0.160thrd3=0.089 divg=0.095 Nucleic acid sequence=SEQ ID NO: 623 Amino acidsequence=SEQ ID NO: 1323; Antibody Heavy Chain No:28564 germ=IGHV1-2*02seqid1=52.9 seqid3=49.2 thrd1=0.163 thrd3=0.124 divg=0.095 Nucleic acidsequence=SEQ ID NO: 624 Amino acid sequence=SEQ ID NO: 1324; AntibodyHeavy Chain No:52736 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181thrd3=0.193 divg=0.095 Nucleic acid sequence=SEQ ID NO: 625 Amino acidsequence=SEQ ID NO: 1325; Antibody Heavy Chain No:105785 germ=IGHV1-2*02seqid1=53.7 seqid3=52.3 thrd1=0.173 thrd3=0.188 divg=0.095 Nucleic acidsequence=SEQ ID NO: 626 Amino acid sequence=SEQ ID NO: 1326; AntibodyHeavy Chain No:12559 germ=IGHV1-2*02 seqid1=49.6 seqid3=52.3 thrd1=0.160thrd3=0.089 divg=0.095 Nucleic acid sequence=SEQ ID NO: 627 Amino acidsequence=SEQ ID NO: 1327; Antibody Heavy Chain No:126568 germ=IGHV1-2*02seqid1=56.2 seqid3=52.3 thrd1=0.164 thrd3=0.121 divg=0.095 Nucleic acidsequence=SEQ ID NO: 628 Amino acid sequence=SEQ ID NO: 1328; AntibodyHeavy Chain No:93926 germ=IGHV1-2*02 seqid1=49.6 seqid3=52.3 thrd1=0.160thrd3=0.089 divg=0.095 Nucleic acid sequence=SEQ ID NO: 629 Amino acidsequence=SEQ ID NO: 1329; Antibody Heavy Chain No:50697 germ=IGHV1-2*02seqid1=49.6 seqid3=52.3 thrd1=0.160 thrd3=0.089 divg=0.095 Nucleic acidsequence=SEQ ID NO: 630 Amino acid sequence=SEQ ID NO: 1330; AntibodyHeavy Chain No:44028 germ=IGHV1-2*02 seqid1=53.7 seqid3=51.5 thrd1=0.148thrd3=0.120 divg=0.095 Nucleic acid sequence=SEQ ID NO: 631 Amino acidsequence=SEQ ID NO: 1331; Antibody Heavy Chain No:43578 germ=IGHV1-2*02seqid1=55.4 seqid3=51.5 thrd1=0.171 thrd3=0.190 divg=0.095 Nucleic acidsequence=SEQ ID NO: 632 Amino acid sequence=SEQ ID NO: 1332; AntibodyHeavy Chain No:41655 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5 thrd1=0.180thrd3=0.193 divg=0.095 Nucleic acid sequence=SEQ ID NO: 633 Amino acidsequence=SEQ ID NO: 1333; Antibody Heavy Chain No:74815 germ=IGHV1-2*02seqid1=55.4 seqid3=50.8 thrd1=0.173 thrd3=0.193 divg=0.095 Nucleic acidsequence=SEQ ID NO: 634 Amino acid sequence=SEQ ID NO: 1334; AntibodyHeavy Chain No:8194 germ=IGHV1-2*02 seqid1=38.0 seqid3=39.2 thrd1=0.235thrd3=0.238 divg=0.095 Nucleic acid sequence=SEQ ID NO: 635 Amino acidsequence=SEQ ID NO: 1335; Antibody Heavy Chain No:141541 germ=IGHV1-2*02seqid1=50.4 seqid3=50.8 thrd1=0.167 thrd3=0.213 divg=0.095 Nucleic acidsequence=SEQ ID NO: 636 Amino acid sequence=SEQ ID NO: 1336; AntibodyHeavy Chain No:24104 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2 thrd1=0.177thrd3=0.145 divg=0.095 Nucleic acid sequence=SEQ ID NO: 637 Amino acidsequence=SEQ ID NO: 1337; Antibody Heavy Chain No:38838 germ=IGHV1-2*02seqid1=55.4 seqid3=52.3 thrd1=0.166 thrd3=0.121 divg=0.095 Nucleic acidsequence=SEQ ID NO: 638 Amino acid sequence=SEQ ID NO: 1338; AntibodyHeavy Chain No:17924 germ=IGHV1-2*02 seqid1=52.1 seqid3=46.9 thrd1=0.227thrd3=0.194 divg=0.095 Nucleic acid sequence=SEQ ID NO: 639 Amino acidsequence=SEQ ID NO: 1339; Antibody Heavy Chain No:13341 germ=IGHV1-2*02seqid1=52.9 seqid3=47.7 thrd1=0.122 thrd3=0.145 divg=0.095 Nucleic acidsequence=SEQ ID NO: 640 Amino acid sequence=SEQ ID NO: 1340; AntibodyHeavy Chain No:35429 germ=IGHV1-2*02 seqid1=53.7 seqid3=52.3 thrd1=0.173thrd3=0.188 divg=0.095 Nucleic acid sequence=SEQ ID NO: 641 Amino acidsequence=SEQ ID NO: 1341; Antibody Heavy Chain No:34820 germ=IGHV1-2*02seqid1=55.4 seqid3=51.5 thrd1=0.178 thrd3=0.133 divg=0.095 Nucleic acidsequence=SEQ ID NO: 642 Amino acid sequence=SEQ ID NO: 1342; AntibodyHeavy Chain No:65892 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.167thrd3=0.122 divg=0.095 Nucleic acid sequence=SEQ ID NO: 643 Amino acidsequence=SEQ ID NO: 1343; Antibody Heavy Chain No:51501 germ=IGHV1-2*02seqid1=51.2 seqid3=47.7 thrd1=0.144 thrd3=0.104 divg=0.095 Nucleic acidsequence=SEQ ID NO: 644 Amino acid sequence=SEQ ID NO: 1344; AntibodyHeavy Chain No:98150 germ=IGHV1-2*02 seqid1=53.7 seqid3=45.4 thrd1=0.117thrd3=0.140 divg=0.095 Nucleic acid sequence=SEQ ID NO: 645 Amino acidsequence=SEQ ID NO: 1345; Antibody Heavy Chain No:12243 germ=IGHV1-2*02seqid1=48.8 seqid3=49.2 thrd1=0.206 thrd3=0.139 divg=0.095 Nucleic acidsequence=SEQ ID NO: 646 Amino acid sequence=SEQ ID NO: 1346; AntibodyHeavy Chain No:77718 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.178thrd3=0.133 divg=0.095 Nucleic acid sequence=SEQ ID NO: 647 Amino acidsequence=SEQ ID NO: 1347; Antibody Heavy Chain No:32593 germ=IGHV1-2*02seqid1=52.1 seqid3=46.9 thrd1=0.122 thrd3=0.145 divg=0.095 Nucleic acidsequence=SEQ ID NO: 648 Amino acid sequence=SEQ ID NO: 1348; AntibodyHeavy Chain No:32228 germ=IGHV1-2*02 seqid1=55.4 seqid3=50.8 thrd1=0.173thrd3=0.193 divg=0.095 Nucleic acid sequence=SEQ ID NO: 649 Amino acidsequence=SEQ ID NO: 1349; Antibody Heavy Chain No:8376 germ=IGHV1-2*02seqid1=49.6 seqid3=50.0 thrd1=0.163 thrd3=0.205 divg=0.091 Nucleic acidsequence=SEQ ID NO: 650 Amino acid sequence=SEQ ID NO: 1350; AntibodyHeavy Chain No:81308 germ=IGHV1-2*02 seqid1=50.4 seqid3=49.2 thrd1=0.157thrd3=0.145 divg=0.091 Nucleic acid sequence=SEQ ID NO: 651 Amino acidsequence=SEQ ID NO: 1351; Antibody Heavy Chain No:11518 germ=IGHV1-2*02seqid1=48.8 seqid3=48.5 thrd1=0.126 thrd3=0.141 divg=0.091 Nucleic acidsequence=SEQ ID NO: 652 Amino acid sequence=SEQ ID NO: 1352; AntibodyHeavy Chain No:73361 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181thrd3=0.134 divg=0.091 Nucleic acid sequence=SEQ ID NO: 653 Amino acidsequence=SEQ ID NO: 1353; Antibody Heavy Chain No:32234 germ=IGHV1-2*02seqid1=56.2 seqid3=52.3 thrd1=0.164 thrd3=0.121 divg=0.091 Nucleic acidsequence=SEQ ID NO: 654 Amino acid sequence=SEQ ID NO: 1354; AntibodyHeavy Chain No:22598 germ=IGHV1-2*02 seqid1=51.2 seqid3=49.2 thrd1=0.218thrd3=0.142 divg=0.091 Nucleic acid sequence=SEQ ID NO: 655 Amino acidsequence=SEQ ID NO: 1355; Antibody Heavy Chain No:82491 germ=IGHV1-2*02seqid1=55.4 seqid3=50.8 thrd1=0.209 thrd3=0.100 divg=0.091 Nucleic acidsequence=SEQ ID NO: 656 Amino acid sequence=SEQ ID NO: 1356; AntibodyHeavy Chain No:22197 germ=IGHV1-2*02 seqid1=50.4 seqid3=46.9 thrd1=0.171thrd3=0.177 divg=0.091 Nucleic acid sequence=SEQ ID NO: 657 Amino acidsequence=SEQ ID NO: 1357; Antibody Heavy Chain No:22110 germ=IGHV1-2*02seqid1=52.9 seqid3=49.2 thrd1=0.198 thrd3=0.154 divg=0.091 Nucleic acidsequence=SEQ ID NO: 658 Amino acid sequence=SEQ ID NO: 1358; AntibodyHeavy Chain No:10233 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.176thrd3=0.131 divg=0.091 Nucleic acid sequence=SEQ ID NO: 659 Amino acidsequence=SEQ ID NO: 1359; Antibody Heavy Chain No:59890 germ=IGHV1-2*02seqid1=55.4 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.091 Nucleic acidsequence=SEQ ID NO: 660 Amino acid sequence=SEQ ID NO: 1360; AntibodyHeavy Chain No:72262 germ=IGHV1-2*02 seqid1=38.0 seqid3=43.1 thrd1=0.331thrd3=0.259 divg=0.091 Nucleic acid sequence=SEQ ID NO: 661 Amino acidsequence=SEQ ID NO: 1361; Antibody Heavy Chain No:101709 germ=IGHV1-2*02seqid1=52.1 seqid3=47.7 thrd1=0.143 thrd3=0.103 divg=0.091 Nucleic acidsequence=SEQ ID NO: 662 Amino acid sequence=SEQ ID NO: 1362; AntibodyHeavy Chain No:64491 germ=IGHV1-2*02 seqid1=56.2 seqid3=52.3 thrd1=0.164thrd3=0.121 divg=0.091 Nucleic acid sequence=SEQ ID NO: 663 Amino acidsequence=SEQ ID NO: 1363; Antibody Heavy Chain No:68980 germ=IGHV1-2*02seqid1=56.2 seqid3=51.5 thrd1=0.158 thrd3=0.138 divg=0.091 Nucleic acidsequence=SEQ ID NO: 664 Amino acid sequence=SEQ ID NO: 1364; AntibodyHeavy Chain No:106681 germ=IGHV1-2*02 seqid1=53.7 seqid3=51.5thrd1=0.148 thrd3=0.120 divg=0.091 Nucleic acid sequence=SEQ ID NO: 665Amino acid sequence=SEQ ID NO: 1365; Antibody Heavy Chain No:9252germ=IGHV1-2*02 seqid1=52.1 seqid3=49.2 thrd1=0.161 thrd3=0.151divg=0.091 Nucleic acid sequence=SEQ ID NO: 666 Amino acid sequence=SEQID NO: 1366; Antibody Heavy Chain No:9061 germ=IGHV1-2*02 seqid1=55.4seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.091 Nucleic acid sequence=SEQID NO: 667 Amino acid sequence=SEQ ID NO: 1367; Antibody Heavy ChainNo:47729 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.176 thrd3=0.131divg=0.091 Nucleic acid sequence=SEQ ID NO: 668 Amino acid sequence=SEQID NO: 1368; Antibody Heavy Chain No:61890 germ=IGHV1-2*02 seqid1=51.2seqid3=49.2 thrd1=0.147 thrd3=0.119 divg=0.091 Nucleic acid sequence=SEQID NO: 669 Amino acid sequence=SEQ ID NO: 1369; Antibody Heavy ChainNo:44167 germ=IGHV1-2*02 seqid1=53.7 seqid3=51.5 thrd1=0.148 thrd3=0.120divg=0.091 Nucleic acid sequence=SEQ ID NO: 670 Amino acid sequence=SEQID NO: 1370; Antibody Heavy Chain No:88530 germ=IGHV1-2*02 seqid1=33.1seqid3=35.4 thrd1=0.208 thrd3=0.361 divg=0.091 Nucleic acid sequence=SEQID NO: 671 Amino acid sequence=SEQ ID NO: 1371; Antibody Heavy ChainNo:113877 germ=IGHV1-2*02 seqid1=41.3 seqid3=42.3 thrd1=0.277thrd3=0.259 divg=0.091 Nucleic acid sequence=SEQ ID NO: 672 Amino acidsequence=SEQ ID NO: 1372; Antibody Heavy Chain No:14850 germ=IGHV1-2*02seqid1=54.5 seqid3=51.5 thrd1=0.181 thrd3=0.134 divg=0.091 Nucleic acidsequence=SEQ ID NO: 673 Amino acid sequence=SEQ ID NO: 1373; AntibodyHeavy Chain No:35041 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181thrd3=0.134 divg=0.091 Nucleic acid sequence=SEQ ID NO: 674 Amino acidsequence=SEQ ID NO: 1374; Antibody Heavy Chain No:33864 germ=IGHV1-2*02seqid1=56.2 seqid3=52.3 thrd1=0.164 thrd3=0.121 divg=0.091 Nucleic acidsequence=SEQ ID NO: 675 Amino acid sequence=SEQ ID NO: 1375; AntibodyHeavy Chain No:10721 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181thrd3=0.134 divg=0.091 Nucleic acid sequence=SEQ ID NO: 676 Amino acidsequence=SEQ ID NO: 1376; Antibody Heavy Chain No:100562 germ=IGHV1-2*02seqid1=55.4 seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.091 Nucleic acidsequence=SEQ ID NO: 677 Amino acid sequence=SEQ ID NO: 1377; AntibodyHeavy Chain No:54476 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.181thrd3=0.134 divg=0.091 Nucleic acid sequence=SEQ ID NO: 678 Amino acidsequence=SEQ ID NO: 1378; Antibody Heavy Chain No:22270 germ=IGHV1-2*02seqid1=52.1 seqid3=49.2 thrd1=0.176 thrd3=0.135 divg=0.091 Nucleic acidsequence=SEQ ID NO: 679 Amino acid sequence=SEQ ID NO: 1379; AntibodyHeavy Chain No:115874 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3thrd1=0.176 thrd3=0.131 divg=0.091 Nucleic acid sequence=SEQ ID NO: 680Amino acid sequence=SEQ ID NO: 1380; Antibody Heavy Chain No:87027germ=IGHV1-2*02 seqid1=52.1 seqid3=48.5 thrd1=0.204 thrd3=0.144divg=0.091 Nucleic acid sequence=SEQ ID NO: 681 Amino acid sequence=SEQID NO: 1381; Antibody Heavy Chain No:35015 germ=IGHV1-2*02 seqid1=55.4seqid3=52.3 thrd1=0.176 thrd3=0.131 divg=0.091 Nucleic acid sequence=SEQID NO: 682 Amino acid sequence=SEQ ID NO: 1382; Antibody Heavy ChainNo:148767 germ=IGHV1-2*02 seqid1=55.4 seqid3=52.3 thrd1=0.176thrd3=0.131 divg=0.091 Nucleic acid sequence=SEQ ID NO: 683 Amino acidsequence=SEQ ID NO: 1383; Antibody Heavy Chain No:28267 germ=IGHV1-2*02seqid1=50.4 seqid3=46.2 thrd1=0.195 thrd3=0.213 divg=0.088 Nucleic acidsequence=SEQ ID NO: 684 Amino acid sequence=SEQ ID NO: 1384; AntibodyHeavy Chain No:63967 germ=IGHV1-2*02 seqid1=48.8 seqid3=46.9 thrd1=0.193thrd3=0.176 divg=0.088 Nucleic acid sequence=SEQ ID NO: 685 Amino acidsequence=SEQ ID NO: 1385; Antibody Heavy Chain No:23404 germ=IGHV1-2*02seqid1=50.4 seqid3=50.8 thrd1=0.146 thrd3=0.134 divg=0.088 Nucleic acidsequence=SEQ ID NO: 686 Amino acid sequence=SEQ ID NO: 1386; AntibodyHeavy Chain No:137385 germ=IGHV1-2*02 seqid1=52.9 seqid3=50.8thrd1=0.188 thrd3=0.184 divg=0.088 Nucleic acid sequence=SEQ ID NO: 687Amino acid sequence=SEQ ID NO: 1387; Antibody Heavy Chain No:24631germ=IGHV1-2*02 seqid1=54.5 seqid3=53.1 thrd1=0.147 thrd3=0.136divg=0.088 Nucleic acid sequence=SEQ ID NO: 688 Amino acid sequence=SEQID NO: 1388; Antibody Heavy Chain No:79562 germ=IGHV1-2*02 seqid1=51.2seqid3=50.8 thrd1=0.169 thrd3=0.131 divg=0.088 Nucleic acid sequence=SEQID NO: 689 Amino acid sequence=SEQ ID NO: 1389; Antibody Heavy ChainNo:26114 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.169 thrd3=0.131divg=0.088 Nucleic acid sequence=SEQ ID NO: 690 Amino acid sequence=SEQID NO: 1390; Antibody Heavy Chain No:66679 germ=IGHV1-2*02 seqid1=53.7seqid3=50.8 thrd1=0.169 thrd3=0.162 divg=0.088 Nucleic acid sequence=SEQID NO: 691 Amino acid sequence=SEQ ID NO: 1391; Antibody Heavy ChainNo:65060 germ=IGHV1-2*02 seqid1=54.5 seqid3=53.1 thrd1=0.147 thrd3=0.136divg=0.088 Nucleic acid sequence=SEQ ID NO: 692 Amino acid sequence=SEQID NO: 1392; Antibody Heavy Chain No:21715 germ=IGHV1-2*02 seqid1=55.4seqid3=53.8 thrd1=0.243 thrd3=0.131 divg=0.088 Nucleic acid sequence=SEQID NO: 693 Amino acid sequence=SEQ ID NO: 1393; Antibody Heavy ChainNo:81335 germ=IGHV1-2*02 seqid1=52.1 seqid3=48.5 thrd1=0.139 thrd3=0.109divg=0.088 Nucleic acid sequence=SEQ ID NO: 694 Amino acid sequence=SEQID NO: 1394; Antibody Heavy Chain No:19816 germ=IGHV1-2*02 seqid1=54.5seqid3=53.1 thrd1=0.147 thrd3=0.136 divg=0.088 Nucleic acid sequence=SEQID NO: 695 Amino acid sequence=SEQ ID NO: 1395; Antibody Heavy ChainNo:21088 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.169 thrd3=0.131divg=0.088 Nucleic acid sequence=SEQ ID NO: 696 Amino acid sequence=SEQID NO: 1396; Antibody Heavy Chain No:20584 germ=IGHV1-2*02 seqid1=54.5seqid3=53.1 thrd1=0.147 thrd3=0.136 divg=0.088 Nucleic acid sequence=SEQID NO: 697 Amino acid sequence=SEQ ID NO: 1397; Antibody Heavy ChainNo:55431 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.169 thrd3=0.131divg=0.088 Nucleic acid sequence=SEQ ID NO: 698 Amino acid sequence=SEQID NO: 1398; Antibody Heavy Chain No:27745 germ=IGHV1-2*02 seqid1=51.2seqid3=51.5 thrd1=0.197 thrd3=0.286 divg=0.088 Nucleic acid sequence=SEQID NO: 699 Amino acid sequence=SEQ ID NO: 1399; Antibody Heavy ChainNo:27991 germ=IGHV1-2*02 seqid1=47.9 seqid3=46.2 thrd1=0.173 thrd3=0.137divg=0.088 Nucleic acid sequence=SEQ ID NO: 700 Amino acid sequence=SEQID NO: 1400; Antibody Heavy Chain No:53541 germ=IGHV1-2*02 seqid1=53.7seqid3=50.8 thrd1=0.169 thrd3=0.162 divg=0.088 Nucleic acid sequence=SEQID NO: 701 Amino acid sequence=SEQ ID NO: 1401; Antibody Heavy ChainNo:15670 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.125 thrd3=0.170divg=0.088 Nucleic acid sequence=SEQ ID NO: 702 Amino acid sequence=SEQID NO: 1402; Antibody Heavy Chain No:46476 germ=IGHV1-2*02 seqid1=55.4seqid3=53.1 thrd1=0.148 thrd3=0.136 divg=0.088 Nucleic acid sequence=SEQID NO: 703 Amino acid sequence=SEQ ID NO: 1403; Antibody Heavy ChainNo:41234 germ=IGHV1-2*02 seqid1=54.5 seqid3=50.8 thrd1=0.162 thrd3=0.120divg=0.088 Nucleic acid sequence=SEQ ID NO: 704 Amino acid sequence=SEQID NO: 1404; Antibody Heavy Chain No:26766 germ=IGHV1-2*02 seqid1=52.1seqid3=48.5 thrd1=0.139 thrd3=0.109 divg=0.088 Nucleic acid sequence=SEQID NO: 705 Amino acid sequence=SEQ ID NO: 1405; Antibody Heavy ChainNo:34607 germ=IGHV1-2*02 seqid1=50.4 seqid3=46.2 thrd1=0.177 thrd3=0.095divg=0.088 Nucleic acid sequence=SEQ ID NO: 706 Amino acid sequence=SEQID NO: 1406; Antibody Heavy Chain No:38262 germ=IGHV1-2*02 seqid1=51.2seqid3=50.0 thrd1=0.153 thrd3=0.176 divg=0.088 Nucleic acid sequence=SEQID NO: 707 Amino acid sequence=SEQ ID NO: 1407; Antibody Heavy ChainNo:2032 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8 thrd1=0.169 thrd3=0.131divg=0.088 Nucleic acid sequence=SEQ ID NO: 708 Amino acid sequence=SEQID NO: 1408; Antibody Heavy Chain No:133700 germ=IGHV1-2*02 seqid1=52.9seqid3=50.0 thrd1=0.195 thrd3=0.162 divg=0.088 Nucleic acid sequence=SEQID NO: 709 Amino acid sequence=SEQ ID NO: 1409; Antibody Heavy ChainNo:36987 germ=IGHV1-2*02 seqid1=54.5 seqid3=53.1 thrd1=0.147 thrd3=0.136divg=0.088 Nucleic acid sequence=SEQ ID NO: 710 Amino acid sequence=SEQID NO: 1410; Antibody Heavy Chain No:36221 germ=IGHV1-2*02 seqid1=48.8seqid3=48.5 thrd1=0.160 thrd3=0.166 divg=0.088 Nucleic acid sequence=SEQID NO: 711 Amino acid sequence=SEQ ID NO: 1411; Antibody Heavy ChainNo:24266 germ=IGHV1-2*02 seqid1=54.5 seqid3=53.1 thrd1=0.147 thrd3=0.136divg=0.088 Nucleic acid sequence=SEQ ID NO: 712 Amino acid sequence=SEQID NO: 1412; Antibody Heavy Chain No:51353 germ=IGHV1-2*02 seqid1=51.2seqid3=50.8 thrd1=0.166 thrd3=0.186 divg=0.088 Nucleic acid sequence=SEQID NO: 713 Amino acid sequence=SEQ ID NO: 1413; Antibody Heavy ChainNo:59427 germ=IGHV1-2*02 seqid1=51.2 seqid3=47.7 thrd1=0.140 thrd3=0.110divg=0.088 Nucleic acid sequence=SEQ ID NO: 714 Amino acid sequence=SEQID NO: 1414; Antibody Heavy Chain No:9160 germ=IGHV1-2*02 seqid1=50.4seqid3=47.7 thrd1=0.165 thrd3=0.138 divg=0.088 Nucleic acid sequence=SEQID NO: 715 Amino acid sequence=SEQ ID NO: 1415; Antibody Heavy ChainNo:113616 germ=IGHV1-2*02 seqid1=29.8 seqid3=36.9 thrd1=0.366thrd3=0.302 divg=0.088 Nucleic acid sequence=SEQ ID NO: 716 Amino acidsequence=SEQ ID NO: 1416; Antibody Heavy Chain No:20929 germ=IGHV1-24*01seqid1=47.9 seqid3=47.7 thrd1=0.203 thrd3=0.175 divg=0.088 Nucleic acidsequence=SEQ ID NO: 717 Amino acid sequence=SEQ ID NO: 1417; AntibodyHeavy Chain No:108561 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5thrd1=0.142 thrd3=0.108 divg=0.084 Nucleic acid sequence=SEQ ID NO: 718Amino acid sequence=SEQ ID NO: 1418; Antibody Heavy Chain No:10295germ=IGHV1-24*01 seqid1=47.9 seqid3=46.9 thrd1=0.215 thrd3=0.184divg=0.084 Nucleic acid sequence=SEQ ID NO: 719 Amino acid sequence=SEQID NO: 1419; Antibody Heavy Chain No:5982 germ=IGHV1-2*02 seqid1=56.2seqid3=50.0 thrd1=0.198 thrd3=0.148 divg=0.084 Nucleic acid sequence=SEQID NO: 720 Amino acid sequence=SEQ ID NO: 1420; Antibody Heavy ChainNo:102928 germ=IGHV1-2*02 seqid1=53.7 seqid3=51.5 thrd1=0.170thrd3=0.182 divg=0.084 Nucleic acid sequence=SEQ ID NO: 721 Amino acidsequence=SEQ ID NO: 1421; Antibody Heavy Chain No:63623 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.171 thrd3=0.177 divg=0.084 Nucleic acidsequence=SEQ ID NO: 722 Amino acid sequence=SEQ ID NO: 1422; AntibodyHeavy Chain No:20898 germ=IGHV1-2*02 seqid1=52.9 seqid3=51.5 thrd1=0.153thrd3=0.159 divg=0.084 Nucleic acid sequence=SEQ ID NO: 723 Amino acidsequence=SEQ ID NO: 1423; Antibody Heavy Chain No:55760 germ=IGHV1-24*01seqid1=47.1 seqid3=47.7 thrd1=0.194 thrd3=0.153 divg=0.084 Nucleic acidsequence=SEQ ID NO: 724 Amino acid sequence=SEQ ID NO: 1424; AntibodyHeavy Chain No:43527 germ=IGHV1-2*02 seqid1=47.9 seqid3=47.7 thrd1=0.143thrd3=0.132 divg=0.084 Nucleic acid sequence=SEQ ID NO: 725 Amino acidsequence=SEQ ID NO: 1425; Antibody Heavy Chain No:82760 germ=IGHV1-2*02seqid1=53.7 seqid3=51.5 thrd1=0.170 thrd3=0.182 divg=0.084 Nucleic acidsequence=SEQ ID NO: 726 Amino acid sequence=SEQ ID NO: 1426; AntibodyHeavy Chain No:46807 germ=IGHV1-2*02 seqid1=53.7 seqid3=52.3 thrd1=0.243thrd3=0.131 divg=0.084 Nucleic acid sequence=SEQ ID NO: 727 Amino acidsequence=SEQ ID NO: 1427; Antibody Heavy Chain No:17702 germ=IGHV1-2*02seqid1=52.9 seqid3=48.5 thrd1=0.129 thrd3=0.165 divg=0.084 Nucleic acidsequence=SEQ ID NO: 728 Amino acid sequence=SEQ ID NO: 1428; AntibodyHeavy Chain No:83355 germ=IGHV1-2*02 seqid1=50.4 seqid3=46.9 thrd1=0.148thrd3=0.127 divg=0.084 Nucleic acid sequence=SEQ ID NO: 729 Amino acidsequence=SEQ ID NO: 1429; Antibody Heavy Chain No:94471 germ=IGHV1-2*02seqid1=52.1 seqid3=50.8 thrd1=0.171 thrd3=0.084 divg=0.084 Nucleic acidsequence=SEQ ID NO: 730 Amino acid sequence=SEQ ID NO: 1430; AntibodyHeavy Chain No:8425 germ=IGHV1-2*02 seqid1=52.9 seqid3=48.5 thrd1=0.170thrd3=0.137 divg=0.084 Nucleic acid sequence=SEQ ID NO: 731 Amino acidsequence=SEQ ID NO: 1431; Antibody Heavy Chain No:54065 germ=IGHV1-2*02seqid1=46.3 seqid3=44.6 thrd1=0.205 thrd3=0.131 divg=0.084 Nucleic acidsequence=SEQ ID NO: 732 Amino acid sequence=SEQ ID NO: 1432; AntibodyHeavy Chain No:154038 germ=IGHV1-2*02 seqid1=51.2 seqid3=48.5thrd1=0.257 thrd3=0.285 divg=0.084 Nucleic acid sequence=SEQ ID NO: 733Amino acid sequence=SEQ ID NO: 1433; Antibody Heavy Chain No:31030germ=IGHV1-2*02 seqid1=52.1 seqid3=47.7 thrd1=0.172 thrd3=0.167divg=0.084 Nucleic acid sequence=SEQ ID NO: 734 Amino acid sequence=SEQID NO: 1434; Antibody Heavy Chain No:6964 germ=IGHV1-2*02 seqid1=51.2seqid3=51.5 thrd1=0.140 thrd3=0.136 divg=0.084 Nucleic acid sequence=SEQID NO: 735 Amino acid sequence=SEQ ID NO: 1435; Antibody Heavy ChainNo:94801 germ=IGHV1-2*02 seqid1=54.5 seqid3=51.5 thrd1=0.167 thrd3=0.209divg=0.084 Nucleic acid sequence=SEQ ID NO: 736 Amino acid sequence=SEQID NO: 1436; Antibody Heavy Chain No:54498 germ=IGHV1-2*02 seqid1=54.5seqid3=52.3 thrd1=0.166 thrd3=0.208 divg=0.084 Nucleic acid sequence=SEQID NO: 737 Amino acid sequence=SEQ ID NO: 1437; Antibody Heavy ChainNo:181191 germ=IGHV1-2*02 seqid1=49.6 seqid3=50.0 thrd1=0.139thrd3=0.123 divg=0.084 Nucleic acid sequence=SEQ ID NO: 738 Amino acidsequence=SEQ ID NO: 1438; Antibody Heavy Chain No:29619 germ=IGHV1-2*02seqid1=50.4 seqid3=48.5 thrd1=0.144 thrd3=0.114 divg=0.084 Nucleic acidsequence=SEQ ID NO: 739 Amino acid sequence=SEQ ID NO: 1439; AntibodyHeavy Chain No:138957 germ=IGHV1-2*02 seqid1=52.1 seqid3=53.8thrd1=0.173 thrd3=0.143 divg=0.084 Nucleic acid sequence=SEQ ID NO: 740Amino acid sequence=SEQ ID NO: 1440; Antibody Heavy Chain No:27733germ=IGHV1-2*02 seqid1=52.9 seqid3=52.3 thrd1=0.137 thrd3=0.219divg=0.081 Nucleic acid sequence=SEQ ID NO: 741 Amino acid sequence=SEQID NO: 1441; Antibody Heavy Chain No:54867 germ=IGHV1-2*02 seqid1=54.5seqid3=50.8 thrd1=0.169 thrd3=0.212 divg=0.081 Nucleic acid sequence=SEQID NO: 742 Amino acid sequence=SEQ ID NO: 1442; Antibody Heavy ChainNo:15769 germ=IGHV1-2*02 seqid1=54.5 seqid3=52.3 thrd1=0.169 thrd3=0.126divg=0.081 Nucleic acid sequence=SEQ ID NO: 743 Amino acid sequence=SEQID NO: 1443; Antibody Heavy Chain No:23105 germ=IGHV1-2*02 seqid1=53.7seqid3=51.5 thrd1=0.171 thrd3=0.123 divg=0.081 Nucleic acid sequence=SEQID NO: 744 Amino acid sequence=SEQ ID NO: 1444; Antibody Heavy ChainNo:61167 germ=IGHV1-2*02 seqid1=53.7 seqid3=50.8 thrd1=0.170 thrd3=0.163divg=0.081 Nucleic acid sequence=SEQ ID NO: 745 Amino acid sequence=SEQID NO: 1445; Antibody Heavy Chain No:57037 germ=IGHV1-2*02 seqid1=54.5seqid3=50.8 thrd1=0.169 thrd3=0.212 divg=0.081 Nucleic acid sequence=SEQID NO: 746 Amino acid sequence=SEQ ID NO: 1446; Antibody Heavy ChainNo:20299 germ=IGHV1-2*02 seqid1=53.7 seqid3=52.3 thrd1=0.217 thrd3=0.149divg=0.081 Nucleic acid sequence=SEQ ID NO: 747 Amino acid sequence=SEQID NO: 1447; Antibody Heavy Chain No:19870 germ=IGHV1-2*02 seqid1=53.7seqid3=52.3 thrd1=0.151 thrd3=0.210 divg=0.081 Nucleic acid sequence=SEQID NO: 748 Amino acid sequence=SEQ ID NO: 1448; Antibody Heavy ChainNo:61564 germ=IGHV1-2*02 seqid1=50.4 seqid3=48.5 thrd1=0.238 thrd3=0.134divg=0.081 Nucleic acid sequence=SEQ ID NO: 749 Amino acid sequence=SEQID NO: 1449; Antibody Heavy Chain No:58649 germ=IGHV1-2*02 seqid1=54.5seqid3=50.8 thrd1=0.169 thrd3=0.212 divg=0.081 Nucleic acid sequence=SEQID NO: 750 Amino acid sequence=SEQ ID NO: 1450; Antibody Heavy ChainNo:106478 germ=IGHV1-2*02 seqid1=49.6 seqid3=48.5 thrd1=0.152thrd3=0.163 divg=0.081 Nucleic acid sequence=SEQ ID NO: 751 Amino acidsequence=SEQ ID NO: 1451; Antibody Heavy Chain No:50598 germ=IGHV1-2*02seqid1=48.8 seqid3=48.5 thrd1=0.202 thrd3=0.106 divg=0.081 Nucleic acidsequence=SEQ ID NO: 752 Amino acid sequence=SEQ ID NO: 1452; AntibodyHeavy Chain No:121214 germ=IGHV1-2*02 seqid1=51.2 seqid3=50.8thrd1=0.203 thrd3=0.199 divg=0.081 Nucleic acid sequence=SEQ ID NO: 753Amino acid sequence=SEQ ID NO: 1453; Antibody Heavy Chain No:61758germ=IGHV1-2*02 seqid1=52.9 seqid3=51.5 thrd1=0.116 thrd3=0.124divg=0.081 Nucleic acid sequence=SEQ ID NO: 754 Amino acid sequence=SEQID NO: 1454; Antibody Heavy Chain No:128584 germ=IGHV1-2*02 seqid1=46.3seqid3=45.4 thrd1=0.153 thrd3=0.159 divg=0.081 Nucleic acid sequence=SEQID NO: 755 Amino acid sequence=SEQ ID NO: 1455; Antibody Heavy ChainNo:16992 germ=IGHV1-2*02 seqid1=52.1 seqid3=48.5 thrd1=0.138 thrd3=0.150divg=0.081 Nucleic acid sequence=SEQ ID NO: 756 Amino acid sequence=SEQID NO: 1456; Antibody Heavy Chain No:42053 germ=IGHV1-2*02 seqid1=52.1seqid3=48.5 thrd1=0.185 thrd3=0.170 divg=0.081 Nucleic acid sequence=SEQID NO: 757 Amino acid sequence=SEQ ID NO: 1457; Antibody Heavy ChainNo:41974 germ=IGHV1-2*02 seqid1=45.5 seqid3=46.2 thrd1=0.169 thrd3=0.220divg=0.081 Nucleic acid sequence=SEQ ID NO: 758 Amino acid sequence=SEQID NO: 1458; Antibody Heavy Chain No:8314 germ=IGHV1-2*02 seqid1=49.6seqid3=47.7 thrd1=0.135 thrd3=0.114 divg=0.081 Nucleic acid sequence=SEQID NO: 759 Amino acid sequence=SEQ ID NO: 1459.

Example 5 Creation of VRC01 and VRC01 like Multimeric Antibodies

As disclosed herein VRC01, a broadly neutralizing human IgG1 monoclonalantibody against HIV, was cloned from human B cells obtained from an HIVinfected donor. VRC01 IgG1 was shown to have very potent neutralizationactivity against more than 90% of HIV isolates from all clades. Thismakes it an attractive therapeutic candidate, and a subject forextensive studies aiming to understand the nature of the antigenicstimuli needed to generate VRC01-like antibodies by infection orimmunization. This example describes the characterization anddevelopment of an IgM antibody carrying the VRC01 V region. We alsocompare its neutralizing activity against HIV with that of theoriginally isolated VRC01 IgG1 (see FIG. 92).

The VRC01 V region was cloned into an expression vector containing theconstant region from the m chain. The IgM was then produced in 293Fcells transiently transfected with this plasmid along with two otherplasmids that encoded the VRC01 k light chain and the human J chain,respectively. The IgM was purified by FPLC using a HiTrap IgM column anda Superose-6 size exclusion column.

Secreted pentameric IgM antibodies carrying the VRC01 V region werepurified to homogeneity by IgM affinity chromatography followed by sizeexclusion chromatography. On a molar basis comparison with theVRC01-IgG, the IgM antibody has increased in vitro neutralizing activityagainst a panel of VRC01-sensitive and resistant HIV virus strains.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

Example 6

Maturation of VRC01-like Antibodies

This example issustrates the nucleic acid and protein sequences ofgermline versions of VRC01-like antibodies. These germline versions ofVRC01-like antibodies are useful, for example, in the development of avaccine or a vaccine evaluation system for HIV-1. VRC01-like antibodies(e.g., VRC01) originated from a germline sequence that “evolved” in vivothrough affinity maturation through repeated exposure of the immunesystem to HIV-1 gp120. Vaccines composed of antigens that are recognizedby immunoglobulin sequences (germline sequences) present in HIV-1 naïvepersons represent a rational starting point for HIV vaccine designes.Multiple germline sequences isolated from the VRC01 donor (Donor 45) aredescribed below:

VRC01 Germline (naïve-like) Rearrangements

Amino acid sequences and encoding DNA:

1. IGHV1-02*02_IGHD3-16*-2_(L14)_IGHJ1*01 (SEQ ID NO: 1460)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtactaggggaaaaaactgtgattacgtttgggacttccagcactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO: 1461)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCTRGKNCDYVWDFQHWGQGTLVTVSS2. IGHV1-02*02_IGHD3-16*02_(L14)_IGHJ4*02 (SEQ ID NO: 1462)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtactaggggaaaaaactgtgattacgtttgggactttgactactggggccagggaaccctggtcaccgtctcctcag(SEQ ID NO: 1463)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCTRGKNCDYVWDFDYWGQGTLVTVSS3. IGHV1-02*02_IGHD3-16*02_(L16)_IGHJ1*01 (SEQ ID NO: 1464)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtactaggggaaaaaactAtgattacgtttgggacttccagcactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO: 1465)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRFTMTRDTSISTAYMELSRLRSDDTAVYYCTRGKNYDYVWDFQHWGQGTLVTVSS4. IGHV1-02*02_IGHD3-16*02_(L18)_IGHJ1*01 (SEQ ID NO: 1466)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtactaggggaaaaaattAtgattacgtttgggacttccagcactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO: 1467)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCTRGKNYDYVWDFQHWGQGTLVTVSS5. IGKV3-11*01_(3′del)_IGKJ2*01 (SEQ ID NO: 1468gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac(SEQ ID NO: 1469)EIVLTQSPATSLSPGERATLSCRASQSVSLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKLEIK 6. IGKV3-11*01_(del6)_IGKJ2*01(SEQ ID NO: 1470)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtggctaCttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1471)HSEIVLTQSPATLSLSPGERATLSCRASQSGYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKLEIK 7. IGKV3-11*01_IGKJ2*01(SEQ ID NO: 1472)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctaCttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1473)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKLEIK 8. IGKV3-11*01_IGKJ2*01_(Long Ins)(SEQ ID NO: 1474)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaaggtccaggtcgaca (SEQ ID NO: 1475)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYEFFGQGTKVQVD 9. IGKV3-NL1_(3′del6)_IGKJ2*01(SEQ ID NO: 1476)gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac(SEQ ID NO: 1477)EIVLTQSPATLSLSPGERATLSCRASQSVSLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYEFFGQGTKLEIK 10. IGKV3-NL1_(del6)_IGKJ2*01(SEQ ID NO: 1478)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtggctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1479)HSEIVLTQSPATLSLSPGERATLSCRASQSGYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYEFFGQGTKLEIK 11. IGKV3-NL1*01_IGKJ2*01(SEQ ID NO: 1480)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1481)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASTRATGIP ARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYEFFGQGTKLEIK12. IGKV3-NL1*01_IGKJ2*01_(LongIns) (SEQ ID NO: 1482)cattcagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatggtgcatccaccagggccactggcatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtcagcagtatgaattttttggccaggggaccaaggtccaggtcgaca (SEQ ID NO: 1483)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASTRATGIP ARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYEFFGQGTKVQVD

VRC03 Germline (naïve-like) Rearrangements

1. IGHV1-02*02_(ins)_IGHD3-22*02_(L9)_IGHJ1*01 (SEQ ID NO: 1484)caggtgcagtggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgactcgacaattatctcaagaccccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgtccggagagggtcctgtggttattgcggagactttccctggcaatactggggccagggcaccctggtcaccgtacctcag(SEQ ID NO: 1485)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVTGPWTRAVDGMDQPQWWHKLCTEVSGQGHHDSQLSQDPRDTSISTAYMELSRLRSDDTAVYYCVRRGSCGYCGDFPWQYWGQGTLVTVSS2. IGHV1-02*02_(ins)_IGHD3-16_(L8)_IGHJ1*01 (SEQ ID NO: 1486)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgactcgacaattatctcaagaccccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgtccggagagggtcctatgattattgcggagactttccctggcaatactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO: 1487)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVTGPWTRAVDGMDQPQWWHKLCTEVSGQGHHDSQLSQDPRDTSISTAYMELSRLRSDDTAVYYCVRRGSYDYCGDFPWQYWGQGTLVTVSS3. IGHV1-02*01_IGHD3-16_(L8)_IGHJ1*01 (SEQ ID NO: 1488)caggtgcagctggtgcagtaggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccagtaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgtccggagagggtcctatgattattgcggagactttccctggcaatactggggccagggcaccctggtcaccgtctcctcag(SEQ ID NO: 1489)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTSTRDTSISTAYMELSRLRSDDTAVYYCVRRGSYDYCGDFPWQYWGQGTLVTVSS4. IGKV3-20*01_(3′del)_IGKJ2*01 (SEQ ID NO: 1490)gaaattgtgagacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcaacaatttgaattttttggccaggggaccaagctggagatcaaac(SEQ ID NO: 1491)EIVLTQSPGTLSLSPGERATLSCRASQSVSSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFEFFGQGTKLEIK 5. IGKV3-20*01_(dEl)_IGKJ2*01(SEQ ID NO: 1492)gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtggcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcaacaatttgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1493)EIVLTQSPGTLSLSPGERATLSCRASQSGSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFEFFGQGTKLEIK 6. IGKV3-20*01_IGKJ2*01(SEQ ID NO: 1494)gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcaacaatttgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1495)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFEFFGQGTKLEIK 7. IGKV3-NL5*01_IGKJ2*01(SEQ ID NO: 1496)gaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtctattactgtcaacaatttgaattttttggccaggggaccaagctggagatcaaac (SEQ ID NO: 1497)EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFEFFGQGTKLEIK 8. IGKV3-NL5*01_(del)_IGKJ2*01(SEQ ID NO: 1498)gaaattgtgttgacgcagtaccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtggcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtctattactgtcaacaatttgaattttttggccaggggaccaagctggagatcaaac(SEQ ID NO: 1499)EIVLTQSPATLSLSPGERATLSCRASQSGSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFEFFGQGTKLEIK 1. VRC 9719 VRC01 germline/IgM:(SEQ ID NO: 1500)MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREKNCDYNWDFEHWGRGTPVIVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY(SEQ ID NO: 1501)atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattcccaggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggccctctggacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagctgagcaggctgagatctgacgacacggccgtgtattactgtgcgagagaaaaaaactgtgattacaattgggacttcgaacactggggccggggcaccccggtcatcgtctcatcagggagtgcatccgccccaacccttttccccctcgtctcctgtgagaattccccgtcggatacgagcagcgtggccgttggctgcctcgcacaggacttccttcccgactccatcactttctcctggaaatacaagaacaactctgacatcagcagcacccggggcttcccatcagtcctgagagggggcaagtacgcagccacctcacaggtgctgctgccttccaaggacgtcatgcagggcacagacgaacacgtggtgtgcaaagtccagcaccccaacggcaacaaagaaaagaacgtgcctcttccagtgattgccgagctgcctcccaaagtgagcgtcttcgtcccaccccgcgacggcttcttcggcaacccccgcaagtccaagctcatctgccaggccacgggtttcagtccccggcagattcaggtgtcctggctgcgcgaggggaagcaggtggggtctggcgtcaccacggaccaggtgcaggagaggccaaagagtctgggcccacgacctacaaggtgaccagcacactgaccatcaaagagagcgactggctcagccagagcatgttcacctgccgcgtggatcacaggggcctgaccttccagcagaatgcgtcctccatgtgtgtccccgatcaagacacagccatccgggtcttcgccatccccccatcctttgccagcatcttcctcaccaagtccaccaagttgacctgcctggtcacagacctgaccacctatgacagcgtgaccatctcctggacccgccagaatggcgaagctgtgaaaacccacaccaacatctccgagagccaccccaatgccactttcagcgccgtgggtgaggccagcatctgcgaggatgactggaattccggggagaggttcacgtgcaccgtgacccacacagacctgccctcgccactgaagcagaccatctcccggcccaagggggtggccctgcacaggcccgatgtctacttgctgccaccagcccgggagcagctgaacctgcgggagtcggccaccatcacgtgcctggtgacgggcttctctcccgcggacgtcttcgtgcagtggatgcagagggggcagcccttgtccccggagaagtatgtgaccagcgccccaatgcctgagccccaggccccaggccggtacttcgcccacagcatcctgaccgtgtccgaagaggaatggaacacgggggagacctacacctgcgtggtggcccatgaggccctgcccaacagggtcaccgagaggaccgtggacaagtccaccggtaaacccaccctgtacaacgtgtccctggtcatgtccgacacagctggcacctgctactga2. VRC 9719 Human J chain: (SEQ ID NO: 1502)MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 1503)atgaagaaccatttgcttttctggggagtcctggcggtttttattaaggctgttcatgtgaaagcccaagaagatgaaaggattgttcttgttgacaacaaatgtaagtgtgcccggattacttccaggatcatccgttcttccgaagatcctaatgaggacattgtggagagaaacatccgaattattgttcctctgaacaacagggagaatatctctgatcccacctcaccattgagaaccagatttgtgtaccatttgtctgacctctgtaaaaaatgtgatcctacagaagtggagctggataatcagatagttactgctacccagagcaatatctgtgatgaagacagtgctacagagacctgctacacttatgacagaaacaagtgctacacagctgtggtcccactcgtatatggtggtgagaccaaaatggtggaaacagccttaaccccagatgcctgctatcctgactaa3. VRC01-H(S239D/I332E): (SEQ ID NO: 1504)MGWSCIILFLVATATGVHSQVQLVQSGGQMKKPGESMRISCRASGYEFIDCTLNWIRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTRGKNCDYNWDFEHWGRGTPVIVSSPSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK(SEQ ID NO: 1505)atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattcccaggtgcagctggtgcagtctggaggtcagatgaagaagcctggcgagtcgatgagaatttcttgtcgggcttctggatatgaatttattgattgtacgctaaattggattcgtctggcccccggaaaaaggcctgagtggatgggatggctgaagcctcgggggggggccgtcaactacgcacgtccacttcagggcagagtgaccatgactcgagacgtttattccgacacagcctttttggagctgcgctcgttgacagtagacgacacggccgtctacttttgtactaggggaaaaaactgtgattacaattgggacttcgaacactggggccggggcaccccggtcatcgtctcatcaccgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccggatgtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccgaggagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

1. An isolated human monoclonal antibody, wherein a heavy chain of theantibody comprises amino acids 26-35 (CDR1), 50-66 (CDR2), and 106-119(CDR3) of SEQ ID NO: 27, and wherein the antibody comprises a lightchain, and wherein the antibody specifically binds gp120 of HIV-1, andwherein the antibody is neutralizing.
 2. The isolated human monoclonalantibody of claim 2, wherein the light chain of the antibody comprisesamino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO:28.
 3. The isolated human monoclonal antibody of claim 1, wherein theheavy chain of the antibody comprises SEQ ID NO:
 27. 4. The isolatedhuman monoclonal antibody of claim 2, wherein the light chain of theantibody comprises SEQ ID NO:
 28. 5. The isolated human monoclonalantibody of claim 1, wherein the antibody is an IgG, IgM or IgA.
 6. Anisolated functional fragment of the isolated human monoclonal antibodyof claim 1, wherein the functional fragment specifically binds gp120 ofHIV-1 and is neutralizing.
 7. The isolated functional fragment of claim6, wherein the fragment is a Fab fragment, a Fab′ fragment, a F(ab)′₂fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fvprotein (dsFv).
 8. The isolated functional fragment of claim 7, whereinthe fragment is a Fab fragment.
 9. The isolated human monoclonalantibody of claim 1, or a functional fragment thereof, wherein theantibody is labeled.
 10. The isolated human monoclonal antibody orfunctional fragment of claim 9, wherein the label is a fluorescent,enzymatic, or radioactive label.
 11. A composition comprising theantibody of claim 1, or a functional fragment thereof that specificallybinds gp120, and a pharmaceutically acceptable carrier.
 12. An isolatednucleic acid molecule encoding the human monoclonal antibody of claim 1,or a functional fragment thereof.
 13. The isolated nucleic acid moleculeof claim 12 comprising at least one of one of the nucleic acid sequencesset forth as SEQ ID NOs: 33-34.
 14. The isolated nucleic acid moleculeof claim 12, operably linked to a promoter.
 15. The isolated humanmonoclonal antibody of claim 1, wherein the heavy chain of the antibodycomprises amino acids 26-35 (CDR1), 50-66 (CDR2), and 106-119 (CDR3) ofSEQ ID NO: 27; and wherein the light chain of the antibody comprisesamino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92 (CDR3) of SEQ ID NO:28.
 16. An isolated host cell transformed with the nucleic acid moleculeof claim
 12. 17. The isolated nucleic acid molecule of claim 12,encoding a heavy chain and a light chain of an antibody, wherein (a) theheavy chain of the antibody comprises amino acids 26-35 (CDR1), 50-66(CDR2), and 106-119 (CDR3) of SEQ ID NO: 27 and the light chain of theantibody comprises amino acids 24-33 (CDR1), 49-55 (CDR2), and 88-92(CDR3) of SEQ ID NO: 28; (b) the heavy chain of the antibody comprisesamino acids 26-35 (CDR1), 50-66 (CDR2), and 106-119 (CDR3) of SEQ ID NO:27 and the light chain of the antibody comprises SEQ ID NO: 28; (c) theheavy chain of the antibody comprises SEQ ID NO: 27 and the light chainof the antibody comprises amino acids 24-33 (CDR1), 49-55 (CDR2), and88-92 (CDR3) of SEQ ID NO: 28; or (d) the heavy chain of the antibodycomprises SEQ ID NO: 27 and the light chain of the antibody comprisesSEQ ID NO: 28, wherein the encoded antibody specifically binds gp120 ofHIV-1, and wherein the encoded antibody is neutralizing.
 18. Anexpression vector encoding the nucleic acid molecule of claim
 1. 19. Theexpression vector of claim 18, encoding an immunoadhesin.
 20. Theexpression vector of claim 18, wherein the antibody is an IgA.
 21. Theexpression vector of claim 18, wherein the expression vector comprises apromoter and an enhancer, and wherein the promoter is a cytomegaloviruspromoter and/or the enhancer is a cytomegalovirus enhancer.
 22. Theexpression vector of claim 18, comprising RNA splicing donor sites, RNAsplicing acceptor sites and/or internal ribosomal binding sequences. 23.The expression vector of claim 18, wherein the heavy chain of theantibody and the light chain of the antibody are expressed as a fusionpolypeptide following the introduction of the expression vector in ahost cell.
 24. The expression vector of claim 23, comprising a nucleicacid sequence encoding a furin cleavage site between the nucleic acidsequence encoding the heavy chain of the antibody and the nucleic acidsequence encoding the light chain of the antibody.
 25. The expressionvector of claim 18, wherein the vector is expressed in Lactobacillus.26. The expression vector of claim 25, comprising a leader sequence thatis expressed in Lactobacillus.
 27. The expression vector of claim 15,encoding a selectable marker.
 28. The expression vector of claim 22,wherein the RNA splicing donor sites are HTLV-1 or CMV RNA splicingdonor sites.
 29. The expression vector of claim 28, wherein the RNAsplicing acceptor sites are HTLV-1 or CMV RNA splicing acceptor sites.30. A method of detecting a human immunodeficiency virus (HIV)-1infection in a subject comprising: contacting a biological sample fromthe subject with at least one isolated human monoclonal antibody ofclaim 1, or a functional fragment thereof that specifically binds gp120and is neutralizing; and detecting antibody bound to the sample, whereinthe presence of antibody bound to the sample indicates that the subjecthas an HIV-1 infection.
 31. The method of claim 30, wherein the isolatedhuman monoclonal antibody is directly labeled.
 32. The method of claim30, further comprising: contacting the sample with a second antibodythat specifically binds the isolated human monoclonal antibody; anddetecting the binding of the second antibody, wherein an increase inbinding of the second antibody to the sample as compared to binding ofthe second antibody to a control sample detects the presence of an HIV-1infection the subject.
 33. A method for preventing or treating an humanimmunodeficiency virus (HIV)-1 infection in a subject, comprisingadministering to the subject a therapeutically effective amount of theantibody of claim 1, or a functional fragment thereof that specificallybinds gp120 and is neutralizing, thereby preventing or treating theHIV-1 infection.
 34. The method of claim 33, wherein the method is amethod for treating an HIV-1 infection, and wherein the subject hasacquired immune deficiency syndrome (AIDS).
 35. The method of claim 33,further comprising administering to the subject an anti-viral agent. 36.The method of claim 33, further comprising measuring HIV-1 viral titerin the subject.
 37. A method for testing a potential vaccine, comprisingcontacting the potential vaccine with an antibody of claim 1, or afunctional fragment thereof that specifically binds gp120 and isneutralizing; and detecting the binding of the antibody to an immunogenin the potential vaccine, thereby identifying the potential vaccine asbeing of clinical interest.